Cloning and functional analysis of the BrCUC2 gene in Brassica rapa L.

The CUP-SHAPED COTYLEDON2 (CUC2) gene plays an important role in the formation of apical meristem and organ edges in plants. The apical meristematic tissue of Brassica rapa (B. rapa) is associated with cold resistance, however, the role of the CUC2 gene in cold resistance of B.rapa is unclear. In this study, we used bioinformatics software to analyze the structure of BrCUC2 gene, real-time fluorescence quantitative PCR to detect the expression level of BrCUC2, constructed transgenic Arabidopsis thaliana by the flower dipping method and subcellular localization for functional validation. The results showed that, we isolated a 1104 bp open reading frame of BrCUC2 from the winter B. rapa cultivar 'Longyou 7'. The BrCUC2 contains a highly conserved domain belonging to the NAM superfamily. Its homologus CUC genes contain similar conserved motifs and are closely related to Brassica oleracea (B.oleracea), and the N-terminal of amino acid sequence contains NAC domain. BrCUC2 protein was localized in the nucleus and self-activation tests showed that pGBKT7-BrCUC2 had self-activation. Tissue-specific expression analysis and promoter ß-Glucuronidase (GUS) activity showed that BrCUC2 had high expression levels in B. rapa growth points and A. thaliana leaf edges, stems and growth points. After low-temperature stress, BrCUC2 showed greater expression in 'Longyou 7,' which presents strong cold resistance and concave growth points, than in 'Longyou 99,' which presents weak cold resistance and protruding growth points. BrCUC2 promoter contains multiple elements related to stress responses. BrCUC2 overexpression revealed that the phenotype did not differ from that of the wild type during the seedling stage but showed weak growth and a dwarf phenotype during the flowering and mature stages. After low-temperature treatment, the physiological indexes and survival rate of BrCUC2-overexpression lines of Arabidopsis thaliana (A. thaliana) were better than those of the wild type within 12 h, although differences were not observed after 24 h. These results showed that BrCUC2 improved the low-temperature tolerance of transgenic A. thaliana within a short time. It can provide a foundation for the study of cold resistance in winter B. rapa.

Corrigendum: Transcriptomic insights into root development and overwintering transcriptional memory of Brassica rapa L. grown in the field.

[This corrects the article DOI: 10.3389/fpls.2022.900708.].

Genome-Wide Identification of C2H2 ZFPs and Functional Analysis of BRZAT12 under Low-Temperature Stress in Winter Rapeseed (Brassica rapa).

Zinc-finger protein (ZFP) transcription factors are among the largest families of transcription factors in plants. They participate in various biological processes such as apoptosis, autophagy, and stemness maintenance and play important roles in regulating plant growth and development and the response to stress. To elucidate the functions of ZFP genes in the low-temperature response of winter (Brassica rapa L.) B. rapa, this study identified 141 members of the C2H2 ZFP gene family from B. rapa, which are heterogeneously distributed on 10 chromosomes and have multiple cis-acting elements related to hormone regulation and abiotic stress of adversity. Most of the genes in this family contain only one CDS, and genes distributed in the same evolutionary branch share mostly the same motifs and are highly conserved in the evolution of cruciferous species. The genes were significantly upregulated in the roots and growth cones of 'Longyou-7', indicating that they play a role in the stress-response process of winter B. rapa. The expression level of the Bra002528 gene was higher in the strongly cold-resistant varieties than in the weakly cold-resistant varieties after low-temperature stress. The survival rate and BrZAT12 gene expression of trans-BrZAT12 Arabidopsis thaliana (Arabidopsis) were significantly higher than those of the wild-type plants at low temperature, and the enzyme activities in vivo were higher than those of the wild-type plants, indicating that the BrZAT12 gene could improve the cold resistance of winter B. rapa. BrZAT12 expression and superoxide dismutase and ascorbate peroxidase enzyme activities were upregulated in winter B. rapa after exogenous ABA treatment. BrZAT12 expression and enzyme activities decreased after the PD98059 treatment, and BrZAT12 expression and enzyme activities were higher than in the PD98059 treatment but lower than in the control after both treatments together. It is speculated that BrZAT12 plays a role in the ABA signaling process in which MAPKK is involved. This study provides a theoretical basis for the resolution of cold-resistance mechanisms in strong winter B. rapa.

Discovery of anti-stroke active substances in Guhong injection based on multi-phenotypic screening of zebrafish.

Ischemic stroke is a leading cause of death worldwide, and it remains an urgent task to develop novel and alternative therapeutic strategies for the disease. We previously reported the positive effects of Guhong injection (GHI), composed of safflower extract and aceglutamide, in promoting functional recovery in ischemic stroke mice. However, the active substances and pharmacological mechanism of GHI is still elusive. Aiming to identify the active anti-stroke components in GHI, here we conducted a multi-phenotypic screening in zebrafish models of phenylhydrazine-induced thrombosis and ponatinib-induced cerebral ischemia. Peripheral and cerebral blood flow was quantified endogenously in erythrocytes fluorescence-labeled thrombosis fish, and baicalein and rutin were identified as major anti-thrombotic substances in GHI. Moreover, using a high-throughput video-tracking system, the effects of locomotion promotion of GHI and its main compounds were analyzed in cerebral ischemia model. Chlorogenic acid and gallic acid showed significant effects in preventing locomotor dyfunctions. Finally, GHI treatment greatly decreased the expression levels of coagulation factors F7 and F2, NF-κB and its mediated proinflammatory cytokines in the fish models. Molecular docking suggested strong affinities between baicalein and F7, and between active substances (baicalein, chlorogenic acid, gallic acid, and rutin) and NF-κB p65. In summary, our findings established a novel drug discovery method based on multi-phenotypic screening of zebrafish, provided endogenous evidences of GHI in preventing thrombus formation and promoting behavioral recovery after cerebral ischemia, and identified baicalein, rutin, chlorogenic acid, and gallic acid as active compounds in the management of ischemic stroke.

Transcriptomic Insights Into Root Development and Overwintering Transcriptional Memory of Brassica rapa L. Grown in the Field.

As the only overwintering oil crop in the north area of China, living through winter is the primary feature of winter rapeseed. Roots are the only survival organ during prolonged cold exposure during winter to guarantee flowering in spring. However, little is known about its root development and overwintering memory mechanism. In this study, root collar tissues (including the shoot apical meristem) of three winter rapeseed varieties with different cold resistance, i.e., Longyou-7 (strong cold tolerance), Tianyou-4 (middle cold tolerance), and Lenox (cold-sensitive), were sampled in the pre-winter period (S1), overwintering periods (S2-S5), and re-greening stage (S6), and were used to identify the root development and overwintering memory mechanisms and seek candidate overwintering memory genes by measuring root collar diameter and RNA sequencing. The results showed that the S1-S2 stages were the significant developmental stages of the roots as root collar diameter increased slowly in the S3-S5 stages, and the roots developed fast in the strong cold resistance variety than in the weak cold resistance variety. Subsequently, the RNA-seq analysis revealed that a total of 37,905, 45,102, and 39,276 differentially expressed genes (DEGs), compared to the S1 stage, were identified in Longyou-7, Tianyou-4, and Lenox, respectively. The function enrichment analysis showed that most of the DEGs are significantly involved in phenylpropanoid biosynthesis, plant hormone signal transduction, MAPK signaling pathway, starch and sucrose metabolism, photosynthesis, amino sugar and nucleotide sugar metabolism, and spliceosome, ribosome, proteasome, and protein processing in endoplasmic reticulum pathways. Furthermore, the phenylpropanoid biosynthesis and plant hormone signal transduction pathways were related to the difference in root development of the three varieties, DEGs involved in photosynthesis and carbohydrate metabolism processes may participate in overwintering memory of Longyou-7 and Tianyou-4, and the spliceosome pathway may contribute to the super winter resistance of Longyou-7. The transcription factor enrichment analysis showed that the WRKY family made up the majority in different stages and may play an important regulatory role in root development and overwintering memory. These results provide a comprehensive insight into winter rapeseed's complex overwintering memory mechanisms. The identified candidate overwintering memory genes may also serve as important genetic resources for breeding to further improve the cold resistance of winter rapeseed.

A Chromosome Level Genome Assembly of a Winter Turnip Rape (Brassica rapa L.) to Explore the Genetic Basis of Cold Tolerance.

Winter rapeseed (Brassica rapa L.) is an important overwintering oilseed crop that is widely planted in northwest China and suffers chronic low temperatures in winter. So the cold stress becomes one of the major constraints that limit its production. The currently existing genomes limit the understanding of the cold-tolerant genetic basis of rapeseed. Here we assembled a high-quality long-read genome of B. rapa "Longyou-7" cultivar, which has a cold-tolerant phenotype, and constructed a graph-based pan-genome to detect the structural variations within homologs of currently reported cold-tolerant related genes in the "Longyou-7" genome, which provides an additional elucidation of the cold-tolerant genetic basis of "Longyou-7" cultivar and promotes the development of cold-tolerant breeding in B. rapa.

Guhong injection promotes post-stroke functional recovery via attenuating cortical inflammation and apoptosis in subacute stage of ischemic stroke.

BACKGROUND: As a leading cause of death and disability, alternative therapies for stroke are still limited by its complicated pathophysiological manifestations. Guhong injection (GHI), consisting of safflower aqueous extract and aceglutamide, has been widely applied for the clinical treatment of cerebrovascular diseases, especially ischemic stroke and post-stroke recovery, in China. Recently, a series of studies have reported the positive effect of GHI against cerebral ischemia/reperfusion injury via targeting various molecular mechanisms. However, questions remain on whether treatment with GHI contributes to better functional recovery after stroke and if so, the potential mechanisms and active substances. PURPOSE: The aim of this work was to explore the potential therapeutic possibilities of GHI for the neurological and behavioral recovery after stroke and to investigate the underlying molecular mechanisms as well as active substances. METHODS: The neural and motor deficits as well as cortical lesions after GHI treatment were investigated in a mouse model of transient ischemic stroke. Based on the substance identification of GHI, network pharmacology combined with an experimental verification method was used to systematically decipher the biological processes and signaling pathways closely related to GHI intervention in response to post-stroke functional outcomes. Subsequently, ingenuity pathway analysis (IPA) analysis was performed to determine the anti-stroke active substances targeting to the hub targets involved in the significant molecular pathways regulated by GHI treatment. RESULTS: Therapeutically, administration of GHI observably ameliorated the post-stroke recovery of neural and locomotor function as well as reduced infarct volume and histopathological damage to the cerebral cortex in subacute stroke mice. According to 26 identified or tentatively characterized substances in GHI, the compound-target-pathway network was built. Bioinformatics analysis suggested that inflammatory and apoptotic pathways were tightly associated with the anti-stroke effect of GHI. Based on protein-protein interaction network analysis, the hub targets (such as NF-κB p65, TNF-α, IL-6, IL-1ß, Bax, Bcl-2, and Caspase-3) involved in inflammation and apoptosis were selected. On the one hand, immunofluorescence and ELISA results showed that GHI (10 ml/kg) treatment obviously reduced NF-κB p65 nuclear translocation as well as decreased the abnormally elevated concentrations of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) in damaged cortex tissues. On the other hand, GHI (10 ml/kg) treatment significantly downregulated the number of TUNEL-positive apoptotic cells in ischemic cortex and effectively restored the abnormal expression of Bax, Bcl-2, and Caspase-3. Based on the results of IPA, hydroxysafflor yellow A, baicalin, scutellarin, gallic acid, syringin, chlorogenic acid, kaempferol, kaempferol-3-O-ß-rutinoside, and rutin acted synergistically on core targets, which could be considered as the active substances of GHI. CONCLUSION: Overall, the current findings showed that the beneficial action of GHI on improving post-stroke functional recovery of subacute stroke mice partly via the modulation of cortical inflammation and apoptosis. These findings not only provide a reliable reference for the clinical application of GHI, but also shed light on a promising alternative therapeutic strategy for ischemic stroke patients.

iTRAQ-based quantitative proteome analysis insights into cold stress of Winter Rapeseed (Brassica rapa L.) grown in the field.

Winter rapeseed (Brassica rapa L.) is a major oilseed crop in Northern China, where its production was severely affected by chilling and freezing stress. However, not much is known about the role of differentially accumulated proteins (DAPs) during the chilling and freezing stress. In this study, isobaric tag for relative and absolute quantification (iTRAQ) technology was performed to identify DAPs under freezing stress. To explore the molecular mechanisms of cold stress tolerance at the cellular and protein levels, the morphological and physiological differences in the shoot apical meristem (SAM) of two winter rapeseed varieties, Longyou 7 (cold-tolerant) and Lenox (cold-sensitive), were explored in field-grown plants. Compared to Lenox, Longyou 7 had a lower SAM height and higher collar diameter. The level of malondialdehyde (MDA) and indole-3-acetic acid (IAA) content was also decreased. Simultaneously, the soluble sugars (SS) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, soluble protein (SP) content, and collar diameter were increased in Longyou 7 as compared to Lenox. A total of 6330 proteins were identified. Among this, 98, 107, 183 and 111 DAPs were expressed in L7 CK/Le CK, L7 d/Le d, Le d/Le CK and L7 d/L7 CK, respectively. Quantitative real-time PCR (RT-qPCR) analysis of the coding genes for seventeen randomly selected DAPs was performed for validation. These DAPs were identified based on gene ontology enrichment analysis, which revealed that glutathione transferase activity, carbohydrate-binding, glutathione binding, metabolic process, and IAA response were closely associated with the cold stress response. In addition, some cold-induced proteins, such as glutathione S-transferase phi 2(GSTF2), might play an essential role during cold acclimation in the SAM of Brassica rapa. The present study provides valuable information on the involvement of DAPs during cold stress responses in Brassica rapa L, and hence could be used for breeding experiments.

Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress.

Winter turnip rape (Brassica rapa L.) is an important overwintering oil crop that is widely planted in northwestern China. It considered to be a good genetic resource for cold-tolerant research because its roots can survive harsh winter conditions. Here, we performed comparative transcriptomics analysis of the roots of two winter turnip rape varieties, Longyou7 (L7, strong cold tolerance) and Tianyou2 (T2, low cold tolerance), under normal condition (CK) and cold stress (CT) condition. A total of 8,366 differentially expressed genes (DEGs) were detected between the two L7 root groups (L7CK_VS_L7CT), and 8,106 DEGs were detected for T2CK_VS_T2CT. Among the DEGs, two ω-3 fatty acid desaturase (FAD3), two delta-9 acyl-lipid desaturase 2 (ADS2), one diacylglycerol kinase (DGK), and one 3-ketoacyl-CoA synthase 2 (KCS2) were differentially expressed in the two varieties and identified to be related to fatty acid synthesis. Four glutamine synthetase cytosolic isozymes (GLN), serine acetyltransferase 1 (SAT1), and serine acetyltransferase 3 (SAT3) were down-regulated under cold stress, while S-adenosylmethionine decarboxylase proenzyme 1 (AMD1) had an up-regulation tendency in response to cold stress in the two samples. Moreover, the delta-1-pyrroline-5-carboxylate synthase (P5CS), δ-ornithine aminotransferase (δ-OAT), alanine-glyoxylate transaminase (AGXT), branched-chain-amino-acid transaminase (ilvE), alpha-aminoadipic semialdehyde synthase (AASS), Tyrosine aminotransferase (TAT) and arginine decarboxylase related to amino acid metabolism were identified in two cultivars variously expressed under cold stress. The above DEGs related to amino acid metabolism were suspected to the reason for amino acids content change. The RNA-seq data were validated by real-time quantitative RT-PCR of 19 randomly selected genes. The findings of our study provide the gene expression profile between two varieties of winter turnip rape, which lay the foundation for a deeper understanding of the highly complex regulatory mechanisms in plants during cold treatment.

Genome-Wide Identification and Analysis of the Ascorbate Peroxidase (APX) Gene Family of Winter Rapeseed (Brassica rapa L.) Under Abiotic Stress.

Winter Brassica rapa (B. rapa) is an important oilseed crop in northern China, but the mechanism of its cold resistance remains unclear. Ascorbate peroxidase (APX) plays important roles in the response of this plant to abiotic stress and in scavenging free radicals. In this study, the roles of APX proteins in the cold response and superoxide metabolism pathways in rapeseed species were investigated, and a comprehensive analysis of phylogeny, chromosome distribution, motif identification, sequence structure, gene duplication, and RNA-seq expression profiles in the APX gene family was conducted. Most BrAPX genes were specifically expressed under cold stress and behaved significantly differently in cold-tolerant and weakly cold-resistant varieties. Quantitative real-time-PCR (qRT-PCR) was also used to verify the differences in expression between these two varieties under cold, freezing, drought and heat stress. The expression of five BrAPX genes was significantly upregulated in growth cones at 3 h of cold stress, while their expression was significantly lower at 24 h than at 3 h. The expression of Bra015403 and Bra003918 was significantly higher in "Longyou-7" growth cones than in other treatments. Five BrAPXs (Bra035235, Bra003918, Bra033040, Bra017120, and Bra031934) were closely associated with abiotic stress responses in B. rapa. These candidate genes may play important roles in the response of B. rapa to low temperature stress and provide new information for the elucidation of the cold resistance mechanism in B. rapa.

Screening and verification of reference genes for analysis of gene expression in winter rapeseed (Brassica rapa L.) under abiotic stress.

Winter rapeseed (Brassica rapa L.) is the main oilseed crop in northern China and can safely overwinter at 35 (i.e., Tianshui, China) to 48 degrees north latitude (i.e., Altai, Heilongjiang, Raohe, and Xinjiang, China). In order to identify stable reference genes to understand the molecular mechanisms of stress tolerance in winter rapeseed, internal reference genes of winter rapeseed under four abiotic stresses were analyzed using GeNorm, NormFinder, BestKeeper, and RefFinder software. The most stable combinations of internal reference genes were ß-actin and SAND in cold-stressed leaves, ß-actin and EF1a in cold-stressed roots, F-box and SAND in high temperature-stressed leaves, and PP2A and RPL in high temperature-stressed roots, SAND and PP2A in NaCl-stressed leaves, RPL and UBC in NaCl-stressed roots, RPL and PP2A in PEG-stressed leaves, and PP2A and RPL in PEG-stressed roots. Expression profiles of PXG3 were used to verify these results. The stable reference genes identified in this study are useful tools for identifying stress-responsive genes to understand the molecular mechanisms of stress tolerance in winter rapeseed.

Ecological and economic benefits of planting winter rapeseed (Brassica rapa L.) in the wind erosion area of northern China.

Winter and early spring wind soil erosion have considerable impacts on ecosystems, human well-being and agricultural production in the low precipitation zones of northern China. Little is known about the impact of growing winter rapeseed on ecological cropping systems and the associated economic benefits in the wind erosion area. To explore the winter rapeseed cover effect, we conducted a field experiment in which we covered the soil with winter rapeseed, winter wheat and wheat stubble at different plant density levels and used the spring bare ground as the control (CK). The effects of wind erosion, the "winter rapeseed + " multiple cropping system, and the economic benefits were compared. There was a large difference in the dry matter, the maximum water absorption, the maximum water storage, the soil evaporation and total wind erosion, the amount of sediment transported in the stratum and the wind erosion modulus. Among them, the mean wind erosion modulus of spring sowing bare land was as high as 490.9 kg·hm-2·h-1, which was 7 and 13 times that of winter wheat and winter rapeseed, respectively. As the wind speed increased from 14 to 22 m·s-1, from a small density to a large density, the mean wind erosion modulus decreased from 68 to 17 kg·hm-2·h-1 for winter rapeseed, and 150 to 31 kg·hm-2·h-1 for winter wheat. Total wind-erosion of sediment transport of CK was 18.6 g·m-2 min-1, which was 16 and 31 times the mean value of winter wheat and winter rapeseed, respectively. "Winter rapeseed + " replanting peanuts, potatoes, rice, seed melons and other crops generally increased the production value by 5-74% compared with wheat and corn intercropping, which was 98-255% higher than the traditional wheat single crop. Our results suggested that the suitable area for planting winter rapeseed in northern China was approximately 3.3 × 106 hm2, and in terms of the best economic and ecological effects, the appropriate density was 5 × 105 plants·hm-2 in northern China. Our results indicated that Chinese winter rapeseed was the best choice for preventing wind erosion and improving ecological and economic benefits in winter and spring in northern China; additionally, winter rapeseed has important impacts on agricultural sustainability in semi-arid and arid climates.

[Safe wintering and economic and ecological benefit of winter rapeseed in dry and cold areas of northern China].

The purpose of this study was to realize the security of safe wintering of winter rapeseed in dry and cold regions of northern China. Experiments were conducted with 18 winter rapeseed (Brassica campestris) varieties at 57 sites from 2008 to 2013 to statistically analyze the wintering rate variation of different varieties in dry and cold regions of northern China. The results showed that, the wintering rate varied from 70% to 90% during the study period in different regions, which had no significant difference between different years and varieties, and had high stability and remarkable economic benefit. With Tianshui as a starting point of winter rapeseed planting, the wintering-safe regions included all Gansu Province , the south of Lasa and Linzhi of Xizang, the east of Minhe of Qinghai, up to Urumqi and Baicheng, and the south of Aletai, Tacheng, the east of Kashi of Xinjiang, it also included the regions along Yellow River eastward to Ningxia, the south of Linhe of Inner Mongolia, the north of Shaanxi, the vicinage of Qixian in Shanxi, Daming in Hebei, Tianjin, Beijing, the north of Weifang of Shandong, the south of Huludao of Liaoning and Yanbian of Jilin. Longyou 6, Longyou 7, Longyou 8 and Longyou 9 were the wintering-safe B. rapa varieties.

Noncovalent assembly of reduced graphene oxide and alkyl-grafted mesoporous silica: an effective drug carrier for near-infrared light-responsive controlled drug release.

In this paper, we report the assembly of reduced graphene oxide (RGO) and mesoporous silica grafted with alkyl chains (MSN-C18) to develop a new class of drug carriers which are able to deliver the loaded drug molecules into living cells upon exposure to near-infrared (NIR) light. This novel drug carrier consists of a structure formed by the noncovalent interaction of RGO caps and alkyl chains on the surface of MSN-C18. The capping of RGO sheets on mesoporous silica effectively blocks the pore mouths in the absence of NIR light. Conversely, and very importantly, the photothermal heating effect of RGO leads to a rapid increase in the local temperature upon exposure to NIR light, resulting in the weakening of the RGO sheet/alkyl chain noncovalent interaction. The RGO sheets will then be removed from the MSN surface, and the pores are uncapped. This uncapping mechanism makes it possible to release the loaded drug molecules upon irradiation with NIR light. In the present study, such a noncovalent assembly was examined by the use of doxorubicin as a model drug for NIR light-responsive intracellular controlled release studies. We believe that this noncovalent assembly will prove to be a promising drug delivery system for cancer therapy in the near future.

Redox-responsive degradable honeycomb manganese oxide nanostructures as effective nanocarriers for intracellular glutathione-triggered drug release.

Redox-responsive degradable honeycomb manganese oxide (hMnO2) nanostructures consisting of some lamellar MnO2 platelets were established as a new class of drug carriers for intracellular glutathione-triggered drug release.

Nanometer-sized manganese oxide-quenched fluorescent oligonucleotides: an effective sensing platform for probing biomolecular interactions.

We propose a novel template-assisted strategy to prepare nanometer-sized manganese oxide (nano-MnO2) by self-assembly of some MnO2 platelets and demonstrate its application as a new class of biosensing platform for probing DNA hybridization and aptamer-target interaction in a homogeneous solution.

Remote-controlled drug release from graphene oxide-capped mesoporous silica to cancer cells by photoinduced pH-jump activation.

Remote light control of drug release enhances our ability to address the complexity of biological systems because of its remarkable spatial/temporal resolution. Here, a new class of remote-controlled release system by incorporating photoacid generator (PAG) into graphene oxide-capped mesoporous silica was designed for delivering drug payloads to cancer cells via photoinduced pH-jump activation. PAG was immobilized on pore wall of the boronic acid-grafted mesoporous silica via strong physical adsorption, and then the nanoparticle was capped with graphene oxide sheet by an acid-labile boroester bond, leading to the formation of nanogated ensemble (MSP-BA-GO). Illuminating with a UV light, PAG generated a pH jump, which induced cleavage of the boroester linkers and thus resulted in the uncapping of pore gates. Moreover, folic acid-modified, doxorubicin (DOX)-loaded MSP-BA-GO (DOX@MSP-BA-GOF) showed selective cell internalization via receptor-mediated endocytosis and subsequent released DOX by the remote illumination. We envisioned that this remote-controlled drug delivery system could find potential applications for cancer therapy.

[Effects of antisense acid invertase gene on reducing sugar and starch contents of potato (Solanum tuberosum L.) tuber stored at low temperature].

Two transgenic potato lines with antisense AcInv gene and the non-transgenic varieties were used to test the reducing sugar and starch contents. The results indicated that the reducing sugar content increased and total starch content decreased in all samples after tubers were stored at 4 degrees C for 40 d. The reducing sugar content in tubers of transgenic line "Anti-AcInv Atlantic" and "Anti-AcInv Gannongshu No.2" were lower about 23% and 18% than those of Atlantic and Gannongshu No.2 (Table 1). The total starch and the amylopectin were also decreased by 1% and 1.3% in "Anti-AcInv Atlantic" and 1.4% and 1.7% in "Anti-AcInv Gannongshu No.2" respectively (Table 2). The proportions of amylose and amylopectin were lower in the transgenic lines than the non-transgenic varieties (Figs. 1 and 2). It was only 0.29 for "Anti-AcInv Atlantic" and 0.38 for "Anti-AcInv Gannongshu No.2". Meanwhile, there are fewer dark blue starch particles in transgenic tuber, which is less than in tuber of the non-transgenic varieties by paraffin-cut section method (Fig. 3). After the tubers were transferred to the storage temperature 15-17 degrees C for 20 d, the reducing sugar contents in tubers of the two transgenic lines were significantly lower than that of non-transgenic varieties. It was 0.23% for "Anti-AcInv Atlantic" and 0.24% for "Anti-AcInv Gannongshu No.2" (Table 1). It is suggested that the trans-antisense AcInv gene in the transgenic potato down-regulates the AcInv gene expression after the tubers were stored under low temperature.