青稞HvBADH1基因的克隆及其转化烟草的初步研究

应用RT-PCR结合RACE技术, 从青稞总RNA中扩增得长度为1 512 bp的甜菜碱醛脱氢酶(BADH)基因cDNA全长编码序列。通过氨基酸同源性比对, 发现该序列的推定表达产物与大麦BADH同工酶BBD2的同源性为98.4%, 而与小麦、玉米和水稻等禾本科作物的BADH同源性分别为97.0%、84.7%和85.1%。将克隆到的青稞cDNA序列命名为HvBADH1, 投递到GenBank, 获得收录号EF492983。HvBADH1可以在原核表达系统TB1-pMAL c2x中正常表达出分子量为54.2 kD的多肽链。将HvBADH1的编码ORF, 插入到添加了植物表达CaMV 35S启动子和Nos polyA终止子调控元件的植物表达载体pCAMBIA1301质粒相应的克隆位点中, 构建了HvBADH1基因的农杆菌植物转化系统LBA4404(pCAM-ba)。进而采用农杆菌介导法, 将HvBADH1基因导入烟草中, 对所获得的潮霉素抗性烟草株系进行PCR、Southern blot和RT-PCR等分子生物学检测, 结果表明, 在得到的2个转基因株系中, HvBADH1基因已整合到受体植物基因组中, 并且可以在mRNA水平上进行转录。

Cloning of HvBADH1 Gene from Hulless Barley and Its Transformation in Nicotiana tabacum

Glycine betaine is a nontoxic osmolyte accumulated in the cytoplasm of salt or drought stressed plants, marine animals, and microorganisms. Betaine aldehyde dehydrogenase (BADH) catalyzes the final step in the synthesis of glycine betaine from choline in many plants. In order to reveal the relationship between the BADH expression and salt stress resistance of hulless bar-ley (Hordeum vulgare L. var. nudum Hook.f.), a 1 512 bp cDNA encoding BADH was cloned from hulless barley using the methods of RT-PCR and RACE. This cDNA encoded a 54.2 kD protein containing 232 amino acid residues and HvBADH1 was designated with the accession number of EF492983 in GenBank. HvBADH1 exhibited the highest homology (98.4%) in amino acid sequence with BBD2 gene encoding an isoenzyme of BADH from barley. It also shared highly homology of 97.0%, 84.7%, and 85.1% with BADH wheat, maize, and rice respectively. The HvBADH1 gene was inserted into pMAL c2x and transformed into E.coli cells (TB1) for expression. The recombinant TB1 (harboring pMAL c2x-HvBADH1) cells and control TB1 (harboring empty pMAL c2x) cells were then induced with IPTG. The results revealed that the recombinant E. coli cells could express a fu-sion protein with molecular weight of 96.3 kD. This fusion protein was fused by maltose binding protein (MBP, about 42.1 kD) and the peptide (about 54.2 kD) encoded by HvBADH1. The ORF of HvBADH1 was inserted between CaMV 35S promoter and NOS polyA in T-DNA region of binary expression vector pCAMBIA1301. The recombinant plasmid, designated as pCAM-ba, was transformed into Agrobacterium tumefaciens LBA4404. The HvBADH1 gene was transformed to tobacco mediated by Agro-bacterium. Two hygromycin B (Hyg) resistant regenerated plant strains were selected. PCR detection and Southern blot analysis indicated that all the Hyg resistant tobacco plants contained the alien BADH gene. RT-PCR analysis showed that HvBADH1 gene normally expressed on the mRNA level in the transgenic tobacco plants. The results suggest that HvBADH1 gene is related with the salt tolerance in hulless barley and can express in transgenic plants.