A cDNA clone encoding a vacuolar H+-pyrophosphatase (V-H+-PPase) was isolated from Hordeum brevisubulatum (Trin.) Link by using RACE method. Sequence analysis revealed that HbVP1 contained 2 319 nucleotides of open reading frame (ORF) and 420 nucleotides of 3′-untranslated region. Its encoding protein consisted of 773 amino acid residues, which includes 14 transmembrane helices. The predicated molecular mass is 80.4 kDa with pI of 4.90. The V-H+-PPases in higher plants shared low identity (40-55%) with those of protozoa, marine alga and archaebacteria. HbVP1 transcripts accumulated abundantly in roots, shoots and seeds, and it was also strongly induced by salt treatment.
【目的】克隆强抗寒性牧草——短芒大麦DREB1(dehydration responsive element binding protein 1)转录因子,分析其生理生化特性,为理想抗逆工程基因的筛选和利用奠定理论基础。【方法】利用RACE-PCR(Rapidamplification of cDNAends-polymerase chain reaction)技术分离短芒大麦DREB1转录因子全长cDNA序列,North-ern杂交和凝胶滞留试验分析其在逆境条件下的表达情况,及其与DRE(dehydration responsive element)元件的结合活性。【结果】从强抗寒性短芒大麦中成功分离了1个新的DREB1类转录因子HbDREB1,该基因全长899 bp,其蛋白序列中含有1个典型的AP2/EREBP DNA结构域及"PKK/RPAGRxKFxETRHP"和"DSAWR"、"LWSY"3个DREB1特征标签序列;序列比对分析表明,HbDREB1与其他植物的DREB1类转录因子的同源性较高。HbDREB1在转录水平上明显受冷胁迫诱导表达,具有结合DRE-顺式作用元件的功能及作为转录因子必备的核定位特性。【结论】HbDREB1基因参与了非生物胁迫信号转导,具有提高植物抗寒性的潜能。
[目的]研究强抗寒性牧草——短芒大麦DREB1(dehydration responsive element binding protein1)基因的功能,为理想抗逆工程基因的筛选和利用奠定理论基础。[方法]利用农杆菌介导法转化烟草叶盘,对转基因烟草进行分子生物学鉴定,并对离子渗漏率、脯氨酸表达量生理生化特性进行测定。[结果]HbDREB1整合入烟草基因组,已在转录水平上表达,转基因烟草在冷胁迫条件下的离子渗漏率明显低于对照组,且其脯氨酸表达量明显增高。[结论]HbDREB1基因具有提高植物抗寒性的潜能。
[Objective] The aim was to study the cloning and sequence analysis of rbcS gene of wild barley under salt stress. [Method] The tender leaf blade of wild barley under salt stress was taken as the experimental material. The primers were designed according to the homology of rbcS gene sequences of wheat and barely in Genbank; then PCR amplification,recovery,ligation,transformation and sequencing of rbcS gene were carried out. [Result] Two rbcS genes including rbcS1 and rbcS2 with the length of 1 252 and 908 bp respectively were cloned from the barely genome. rbcS1 and rbcS2 were both composed by two exons and one intron. The exons length of the two genes was the same of 525 bp,encoding 174 amino acids,and the homology between them was 96%; however,the intron length of rbcS1 and rbcS2 was 448 and 107 bp respectively.
[Objective] The aim was to study the expression of cold resistant gene CAS19 of Gongnong No.2 Medicago sativa L. in tobacco. [Method] A pair of primers was designed according to nucleotide sequences of cold resistant gene CAS19 of M. sativa,and then RT-PCR was used to amplify the protein gene of CAS19,which was then cloned into pMD18-T vector and subcloned into expression vector PBI121. The recombination expression plasmid PBCAS was constructed. And then it was transferred into tobacco genome via Agrobacterium,and Southern-blotting analysis was used for detecting transgenic plants. [Result] CAS19 gene was integrated into the tobacco genome and highly expressed. [Conclusion] This study had provided theoretical basis for further exploring the expression mechanism of cold resistant gene CAS19 in tobacco.
