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Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.
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DNA, Complementary , Pinellia , Virology , Plant Diseases , Virology , Potyvirus , MetabolismABSTRACT
Objective: In this study, the transcriptome profile of the stems from Pinellia ternata treated by 30% polyethylene glycol simulated drought stress was analyzed, the key enzyme genes responded to the drought stress were explored, so as to explore the molecular mechanism of P. ternata sprout tumble responded to the drought stress. Methods: The stem segments of P. ternata under drought stress were used as research materials. The Illumina Hiseq 2500 platform was applied for transcriptome sequencing, and bioinformatics analysis was performed on the transcriptome data. Results: A total of 23.00 Gb clean data was obtained, 37 467 952 and 39 903 362 clean reads were gained from the control groups and the drought stress treatment, respectively, and 100 274 uingenes were assembled by Trinity software. A total of 41 132 unigenes were finally obtained with functional annotations against the Nr, eggNOG, Pfam, Swiss-Prot, KOG, GO, KEGG, and COG databases. Furthermore, 4 052 differentially expressed genes (DEGs) were obtained, in details, 2 080 DEGs were up-regulated while 1 972 DEGs were with down-regulated expression. KEGG Pathway enrichment analysis showed that DEGs were mostly involved in ribosome, carbon metabolism, starch and sucrose metabolism, plant hormone signal transduction and biosynthesis of amino acids process. The expression levels of the genes encoded plant hormone metabolism and signal transduction, redox-related enzymes, heat shock proteins and vacuolar processing enzymes were significantly promoted after drought stress treatment, while most of the DEGs encoded aquaporins were down-regulated. Conclusion: By analyzing the transcriptome profiles of P. ternata under drought stress simulated by PEG, the candidate genes associated with water metabolism, plant hormone and signal transduction, as well as the related responsive proteins were obtained, which could provide abundant genetic resources and theoretical basis for understanding the mechanism of P. ternata sprout tumble caused by drought stress.
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Objective: To clone an agglutinin gene from Pinellia ternata and to analyze its bioinformatics and subcellular location. Methods: Based on the published sequence GU593718.1 from Genbank, P. ternata agglutinin (PTA) was amplified and cloned from genomic DNA of the fresh leaves of P. ternata. The cloned PTA gene was further fused to the plant expression vector pI1300-CaMV35S-GFP to construct pI1300-CaMV35S-PTA-GFP, then transfered into cells of Agrobacterium tumefaciens GV3101. Its transient expression was observed in Nicotiana tabacum. Results: The full length of PTA contained 810 bp with the deduced 269 amino acid residues; It contained one signal peptide, two conversation B-lectin domains and three mannose binding sites; PTA shared 97%, 85%, and 83% identity with the amino acid sequence from PTA, and Pinellia pedatisecta agglutinin (PPA), Pinellia cordata agglutinin (PCA), respectively; The PTA was localized to the plasma membrane; Its registration number is KF154979 in NCBI. Conclusion: It would provide a stable foundation for the study on its effect against fungi, insects, and bacterium.
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Objective: To provide the evidence for the identification and genetic diversity of Pinellia ternata by studying cpDNA non-coding sequences of P. ternata and its related species. Methods: Besides P. cordate and P. pedatisecta, 43 P. ternata samples were collected from the main habitats in China. psbK-psbI and atpF-atpH sequences in leaf genome DNA were cloned by PCR. Comparative analysis was carried out by bioinformatics software. Results: The sequence length of atpF-atpH in P. ternata was 337-342 bp and conservative. The numbers of variable sites and parsimony information sites were only 7 and 1, respectively. The genetic distance was 0-0.024.The length of psbK-psbI was 432-435 bp, with 37 variable sites, including 17 information sites, and the genetic distance was 0-0.069.Cluster analysis was not consistent with the phenotype or the geographical distributions. Conclusion: The discrimination of psbK-psbI is better than that of atpF-atpH, and there are more mutation sites in psbK-psbI sequence among species in P. ternata.
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Objective: The effect on yield and quality with different size tubers of Pinellia ternata from Sichuan was studied under the cultivated condition, in order to provide the theoretical basis for standardized cultivation. Methods: Three different populations of P. ternata from Sichuan were classified according to the diameters as follows: smaller (0.5 cm1.5 cm), and this experiment was performed in a randomized block design. During growth and development period, agronomic traits, such as number of sprouting, inflorescence, and bulblets were censused. After harvesting, main chemcial compositions, growth and proliferation rates were determined. Results: The different sizes tubers of P. ternata showed the same growth rhythm. But there were signifiacnt differences in the average emergence rate, the average ratio of bolting, the average bulbils, individual growth rate, individual proliferation rate among different sizes. Also, alkaloid and inosine contents were signifiacntly different. But β-sitosterol content had no signifiacnt difference. Conclusion: The size of tubers the in 0.5 cm1.5 cm) while good strains breeding and propagation.
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Using the tissue culture technology to achieve the fast propagation of P.ternata and through the photosynthetic characters and yield comparison,two high-yield indivaduals were obtained. The result showed that the coefficent of propagation can get to 1∶6.23 per generation, through 3 month culture the average diameter of the in vitro tuber can get 0.88 cm. P.ternata was a typical shade-plant, of which LCP was only 700?mol?photons/s?m2. Photo-inhibition was observed among all the three leaf-types of P.ternata, and the willow leaf type showed the most obvious photo-inhibition. Both of the light response Pn curve and single tuber weight were significant different among leaf styles. Result for studying the chlorophyll fluorescence parameters showed that all the difference of NPQ, qP and qN among three leaf-types of P.ternata were significant and the difference of potential-photosynthetic productivity among them was significant. The willow leaf style had the strongest light-tolerance capacity.The individuals of T2(peach-leaf type) had the highest Pn and single tuber weight among 11 superior individuals and the individuals of L2(willow-leaf type) showed the strongest light-tolerance capacity. Through 6 month propagation ten thousands in virto tuber of T2 and L2 each were gotten. And after growing in Wenzhou experimental base for one year, the total gross weight of the high-yield individual tuber get to 100kg each.
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Objective To study the quality of Pinellia ternata grown in Funan. Methods The contents of free and hydrolyzed amino acids and ephedrine in the samples from four habitats were determined. The components of methanol and acidic ethanol extracts of the four samples were compared by HPLC. The components of petroleum ether extracts of the four samples were compared by TLC. Results The content of amino acids and ephedrine and the kinds of amino acids of P. ternata grown in Funan were almost the same as those from the other three, the main components of methanol and petroleum ether extracts of P. ternata grown in Funan were the same as those from other three. Some components of acidic ethanol extracts of the four samples were different. Conclusion The quality of P. ternata grown in Funan is as good as that of the other three.
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Objective To discriminate Rhizoma Pinelliae from its relative species by electrophoro-gram of protein.Methods Using isoelectrofocusing(IEF) technology to compare the electrophorogram of tuber of Pinellia ternata produced in different habitats and from its relative plants then determining the pH values.Results The IEF electrophorogram of Rhizoma Pinelliae was stable with eight clear characteristic protein bands and had obvious differences from the electrophorogram of the tubers of its relative plants.Conclusion The IEF electrophoresis of Rhizoma Pinelliae can be used as the distinguishing basis.The PI value of the characteristic protein of P.ternana offers the basic parameter for separation and purification of the protein in Rhizoma Pinelliae.
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Objective Industrialized breeding technology system will be established to achieve the rapid multiplication of Pinellia ternata and to settle the problem of degeneration caused by seeds. MethodsExplant induction and effects of plant growth regulator dosage, illumination, and subculture cycle on fasciculated shoot propagation were studied. ResultsIt is good for the explant induction on medium MS+6-BA (4-5) mg/L+NAA 0.2 mg/L. P.ternata grows well under the circumstance of NAA 0.2 mg/L+6-BA 2 mg/L, double or more shoots. ConclusionIndustrialized breeding technology system of P.ternata can make its propagation rapid and support an effective way for resources preserving and sustainable utilization.
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Objective The DNA fingerprints of cultivated Pinellia ternata collected from different geographic regions were generated by using AFLP markers to find the feasibility in analyzing their genetic diversity, relationship, and germplasm identification. Methods The DNA polymorphism of 51 cultivated germplasm of P. ternata collected from 17 different habitats and four cultivars of P. pedatisecta (outgroup) were detected by AFLP molecular markers. Results The DNA fingerprints of 51 individuals of P. ternata were obviously distinguished by eight pairs of high polymorphic and efficient primer combinations screened from 64 primer combinations. The phylogenetic clustering results revealed that all the tested cultivars were fully differentiated, and individuals from the same regions were mainly clustered together. Moreover, cultivars from East-China, including Zhejiang and Jiangsu Provinces, displayed clear genetic distinction from other regions. The clustering results were strongly supported by Bootstrap test. Conclusion AFLP Markers can be potentially used in analyzing of genetic diversity, relationship, and germplasm identification of this medicinal plant, and the germplasm from regions of East-China, including Zhejiang and Jiangsu Provinces, displays the relative separate genetic characters from other regions.
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Objective To analyze the DNA methylation status of polyploid complex of Pinellia ternata. Methods Methylation sensitive amplified polymorphism(MSAP) technique was carried out to analyze the methylation status of polyploid complex of P.ternata. Thirty-four selective primer amplifications were used to check the status of cytosine methylation DNA samples. Results A total of 7 708 bands were obtained.Among them 5 636 bands,each representing a recognition site cleaved by one or both of the isoschizomers(Hpa Ⅱ and Msp Ⅰ),were amplified.Furthermore,methylation patterns varied among the four polyploids:heptaploid,octoploid,nonuploid,and decaploid.Total and full methylation levels in P.ternata were 54%-58% and 24.1%-24.3%.All types of MSAP patterns detected in the study belonged to two classes,type Ⅰ and Ⅱ. 52.5% of detected bands belonged to Type Ⅰ; Another 47.5% were type Ⅱ,which showed the methylation differences among the four polyploids. Conclusion The results demonstrate DNA methylation events occur in P.ternata and the general methylation levels are higher.
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Objective To find out the proper concentration of ZnSO4?7H2O as foliar fertilizer used for Pinellia ternata, and to provide reference for high yield of P.ternate,effects of zinc on the carbon and nitrogen metabolism in leaves of P.ternata and the yield were studied.Methods Seedlings of P.ternata were employed as the experiment material.At the stage of full seedling,ZnSO4?7H2O of different concentration was sprayed on the leaves,the indexes of carbon and nitrogen metabolism were determined on the day 10,the weight of tubers and bulbils were measured at the sprout tumble stage.Results The yield of P.ternata has an exceedingly significant positive correlation with the contents of soluble sucrose and starch in the leaves,a significant positive correlation with the content of photosynthetic pigment and the aldolase activity,and a negative correlation with the amylase activity.In addition,the yield shows both an exceedingly significant positive correlation whith the nitrate reductase activity and a significant positive correlation with the content of protein.At the stage of full seedling,zinc sprayed on leaves can increase the content of photosynthetic pigment and improve the capability of photosynthesis.Meanwhile,spraying zinc can also promote the accumulation of soluble sucrose and starch in the leaves because of its stimulation on aldolase and inhibition on amylase.Besides,spraying zinc on leaves could improve the activity of nitrate reductase and the content of protein,and finally increase the yield of P.ternata.Conclusion The concentration of 600 mg/L is the best for ZnSO4?7H2O used as foliar fertilizer for P.ternate at the stage of full seedling.