青花菜ProDH基因的克隆及功能鉴定

Cloning and Functional Identification of ProDH Gene from Broccoli

脯氨酸是水分胁迫植物中最常见的渗透保护物质之一,脯氨酸脱氢酶(ProDH)是存在于线粒体内的催化脯氨酸降解的关键酶,构建RNAi表达载体转化青花菜可以抑制脯氨酸分解提高其抵抗干旱胁迫和盐胁迫的能力。本研究利用同源重组和RACE技术克隆青花菜脯氨酸脱氢酶基因(ProDH),以酶切、连接的方法利用载体pFGC1008构建植物RNAi表达载体pFGC-PDHi。序列分析表明,本文首次克隆了1595bp的青花菜脯氨酸脱氢酶基因cDNA全长,该序列编码498个氨基酸;序列比对发现该核酸序列与拟南芥、甘蓝型油菜、芜菁分别有83.26%、89.07%和97.73%的同源性,其氨基酸序列和拟南芥、甘蓝型油菜、芜菁分别有89.78%、91.38%和98.80%的同源性。测序和酶切鉴定表明青花菜ProDH基因RNA干扰表达载体已经构建成功,并将其转化青花菜得到转化株,经PCR检测,转化株均为阳性株,证明干扰载体的T-DNA区已经成功地整合到了青花菜基因组中。研究还发现在外源的L-脯氨酸存在时,转化株的脯氨酸脱氢酶的活性受到了明显的抑制。本研究为青花菜的抗旱育种提供了非常有价值的新种质材料。

Proline is one of the most common compatible osmolytes in water-stressed plants. Proline dehydrogenase （ProDH） localized in mitochondria, which is one of the rate-limiting enzymes in degradation ofproline.Transforming the constructed RNAi expression vector to broccoli can decrease the production of proline and increase the capability of resistance to drought stress salt tolerance. In this research, we cloned the BoProDH gene by using homologous recombination and RACE techniques, then constructed plant RNAi expression vector pFGC-PDHi with pFGC1008 vector by digestion and ligasion methods. The sequence analysis indicated that we firstly cloned the full length cDNA sequence containing 1 595 bp, which coded 498 amino acid residues. Sequence alignment found that the nucleotides of ProDH gene showed a high degree of sequence homology with the A tERD5, BnProDH and BrProDH was 83.26%, 89.07% and 97.73%, respectively and its deduced amino acid sequence displayed the similar rate of amino acid sequence was 89.78%, 91.38% and 98.80%, respectively. The results still demonstrated that RNAi expression vector of ProDH gene had been successfully established after sequencing and enzyme digestion, then which was transformed into broccoli obtaine the transforment plants. The transforment plants were positive by PCR validation, which proved that T-DNA region of interference vector had been successfully integrated into broccoli genome. We still found that the activity of ProDH enzyme in trans genic broccoli plants obviously decreased compare with untransformed plants in response to exogenous L-proline. These results would provide very valuable new germplasm materials for drought-resistant breeding of broccoli.