Identification and expression analysis of NHX gene family in Chinese cabbage / 生物工程学报

Na+/H+ antiporter (NHX) gene subfamily plays an important role in plant response to salt stress. In this study, we identified the NHX gene family members of Chinese cabbage and analyzed the expression patterns of BrNHXs gene in response to abiotic stresses such as high temperature, low temperature, drought and salt stress. The results showed that there were 9 members of the NHX gene family in Chinese cabbage, which were distributed on 6 chromosomes respectively. The number of amino acids was 513-1 154 aa, the relative molecular weight was 56 804.22-127 856.66 kDa, the isoelectric point was 5.35-7.68. Members of BrNHX gene family mainly existed in vacuoles, the gene structure is complete, and the number of exons is 11-22. The secondary structures of the proteins encoded by the NHX gene family in Chinese cabbage had alpha helix, beta turn and random coil, and the alpha helix occurred more frequently. Quantitative real-time PCR (qRT-PCR) analysis showed that the gene family members had different responses to high temperature, low temperature, drought and salt stress, and their expression levels differed significantly in different time periods. BrNHX02 and BrNHX09 had the most significant responses to these four stresses, and their expression levels were significantly up-regulated at 72 h after treatments, which could be used as candidate genes to further verify their functions.

Phylogenetic and expression analysis of SEPALLATA-like gene in Brassica oleracea L. var. acephala / 生物工程学报

The E class MADS-box genes SEPALLATA (SEP)-like play critical roles in angiosperm reproductive growth, especially in floral organ differentiation. To analyze the sequence characteristics and spatio-temporal expression patterns of E-function MADS-box SEP-like genes during kale (Brassica oleracea L. var. acephala) flower development, BroaSEP1/2/3 (GenBank No. KC967957, KC967958, KC967960) homologues, three kale SEP MADS-box gene, were isolated from the kale variety 'Fourteen Line' using Rapid amplification of cDNA ends (RACE). Sequence and phylogenetic analysis indicated that these three SEP genes had a high degree of identity with SEP1, SEP2, SEP3 from Brassica oleracea var. oleracea, Brassica rapa, Raphanus sativus and Brassica napus, respectively. Alignment of the predicted amino acid sequences from these genes, along with previously published subfamily members, demonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins: the MADS domain, intervening (I) domain and keratin-like (K) domain, and the C-terminal domain SEPⅠ and SEP Ⅱ motif. The longest open reading frame deduced from the cDNA sequences of BroaSEP1, BroaSEP2, and BroaSEP3 appeared to be 801 bp, 759 bp, 753 bp in length, respectively, which encoded proteins of 266, 252, and 250 amino acids respectively. Expression analyses using semi-quantitative RT-PCR and quantitative real-time PCR indicate that BroaSEP1/2/3 are specifically expressed in floral buds of kale during flower development process. The expression levels of the three genes are very different at different developmental stages, also in wild type, mutant flower with increased petals, and mutant flower with decreased petals. These different patterns of gene expression maybe cause the flowers to increase or decrease the petal number.

Bioconversion of waste cooking oil glycerol from cabbage extract to lactic acid by Rhizopus microsporus

Abstract Glycerol from spent oil was processed by transesterification for biodiesel production. Although glycerol contains many types of impurities, it can be used as a C-source for lactic acid production by fungi, such as Rhizopus microsporus. In this study, we found that wild type R. microsporus (LTH23) produced more lactic acid than the mutant strains on cabbage glycerol media (CG media). More lactic acid was produced on CG media than on cabbage extract media (C media) by about two-fold in batch fermentation conditions. In addition, we found that lactic acid production in a fed-batch process was also slightly higher than in a batch process. To study the combined effects of pH, urea, and glycerol waste concentration on lactic acid production, a response surface methodology was used. The optimum pH, urea, and glycerol waste concentrations were pH 6.5, 3.75 g/L, and 17 g/L, respectively. The maximum lactic acid production predicted by this equation model was 4.03 g/L.

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgroTM) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34ºC. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel.

Accumulation of lead and cadmium in upper parts of mustard (Brassica juncea) seedlings in response to putrescine.

Lead and cadmium accumulation examined in shoot and leaf tissues of seedlings of mustard (Brassica juncea cv RH-30), at 7th day, treated with either putrescine (1 mM), or ammonium nitrate (10 mM) or IAA (10 microM). These were included in the nutrient medium, containing Pb or Cd (0.1 mM and 2 mM). Metal accumulation was more in shoot than in leaf tissues, which was increased manifold under saline conditions. However, Cd accumulation in tissues was higher than Pb. Chemical (putrescine, ammonium nitrate or IAA) treatment of the seedlings, decreased metal accumulation in leaf (10-20%) and in shoot (40 to 60%) tissues, depending upon external metal levels. Putrescine significantly decreased the metal accumulation and translocation under saline conditions.

Fatty acid synthesis by isolated leucoplasts from developing Brassica seeds: role of glycolytic intermediates as the source of carbon and energy.

Fatty acid synthesis from Na [1-14C] acetate in leucoplasts isolated from developing seeds of Brassica campestris was completely dependent on exogenous supply of ATP. None of the intermediates of glycolysis or pentose phosphate pathway tested could replace ATP in the reaction mixture. In absence of exogenously supplied ATP, maximum activity was obtained with glu-6-P (68%) followed by fru-6-P (50%) and PEP (44%), respectively. With other intermediates as energy sources, the activity ranged from 1 to 38%. In complementary experiments (presence of ATP), none of the metabolites gave activity higher than the ATP control activity. Under optimum conditions for fatty acid synthesis from acetate, Brassica leucoplasts readily utilized labelled glucose as the substrate for fatty acid synthesis. Omission of NADH and NADPH individually from the reaction mixtures containing labelled glucose resulted only in 46 and 20% loss in activity, respectively, compared to the corresponding losses of 56 and 50%, when labelled acetate was used as the substrate. Similarly, deletion of ATP from the reaction mixture containing glucose as the substrate decreased the rate of fatty acid synthesis by about 65%, while the corresponding decrease with acetate as the substrate was 96%. Inclusion of 5 mM cold acetate, pyruvate, malate and glu-6-P in the reaction mixture containing glucose as the labelled substrate reduced label incorporation into fatty acids by 38 to 69%, maximum reduction being observed with pyruvate followed by glu-6-P, acetate and malate, respectively. With labelled acetate as the substrate, maximum reduction in label incorporation was obtained with cold glucose (5 mM) followed by glu-6-P, pyruvate and malate, respectively. The study demonstrated the operation of complete glycolytic pathway in Brassica leucoplasts, allowing the plastids to use glucose as a source of carbon, reducing power and energy for fatty acid synthesis.