鸭梨苯丙氨酸解氨酶基因cDNA片断的克隆与序列分析
摘要
果实的食用品质是决定果实经济价值的重要因素。果实食用品质受多种因素的综合影响,石细胞是影响梨果实品质的重要因素之一。石细胞是由果实发育过程中木质素的沉淀形成,因此木质素的合成直接影响到梨果实中石细胞的分化和发育。苯丙氨酸解氨酶(Phenylalanine Ammonia-Lyase,PAL)是木质素生物合成途径中的第一个限速酶,其表达及丰度直接影响木质素生物合成的整个过程。本研究对鸭梨PAL基因片段进行了克隆与序列分析,以期应用分子生物学的手段找出控制梨石细胞形成的基因,从而为梨品种的改良,及选育石细胞含量少的优质梨品种奠定坚实的理论基础及技术支持。本研究取得的主要结果如下:
1.根据苯丙氨酸解氨酶基因保守序列设计一对引物,以鸭梨果实cDNA为模板扩增得到478 bp的鸭梨苯丙氨酸解氨酶基因cDNA片段(GenBank登录号:GU355673),长度为478 bp,编码159个氨基酸残基。
2.利用RT-PCR技术从鸭梨果实中提取的mRNA中扩增出一个大小为478bp的木质素合成特异表达的PAL基因片段,将其克隆到pUCm-T Vector载体上获得了重组质粒,通过对该重组质粒进行了PCR扩增检测以及序列测序分析,证明我们插入到载体上的片段即是我们所扩增出来的DNA片段,对鸭梨苯丙氨酸解氨酶基因序列进行同源性比对,通过比对,其基因序列与GenBank中已登录的蔷薇科的西洋梨的同源性达99%;与枇杷、桃树、覆盆子的同源性分别为96%、87%和80%。从而证明我们所扩增出来的DNA片段即为PAL基因片段的一部分。
3.应用软件对所克隆的鸭梨苯丙氨酸解氨酶基因序列片段进行了酶切位点的检测,结果显示鸭梨苯丙氨酸解氨酶基因序列片段中含有大量的酶切位点,其中含有常用的限制性内切酶MSEⅠ的识别位点。
4.对所克隆的鸭梨苯丙氨酸解氨酶基因编码的氨基酸进行疏水性的检测,其所编码的氨基酸表现为疏水性。并对所克隆片段的氨基酸组成进行了分析,结果表明,鸭梨苯丙氨酸解氨酶基因推导蛋白中常见的二十种氨基酸均有涉及,数量在3~11之间,其中以Leu含量最高,为11%,以Pro含量为最低,仅占1%。在总共159个氨基酸中,共有疏水性氨基酸64个,占氨基酸总数的40%;亲水性氨基酸占50个,占总数的34%;酸性氨基酸21个,碱性氨基酸24个,分别占总数的12%和14%。
The economic values of the pears depend on the edible quality of the pear fruit. And the stone cell is one of the most factors. So the stone cell of the pear fruit takes the direct effect of the quality. The sediment of lignin comes into being the stone cell.The phenylalanine ammonia- lyase (PAL) which links primary and secondary metabolism by catalyzing the conversion of L-phenylalanine to cinnamic acid, is the first key enzyme in the pathway of plant secondary metabolism. The study which used Yali pear as starting material analyzes the sequence of Yali pear. The major results of this dissertation are as follows:
1. High purity RNA was extracted from Yali pear and used as a template for cloning the Phenylalanine Ammonia- Lyase gene cDNA by RT-PCR with specific primers under gradient temperature. Yali pear Phenylalanine Ammonia-Lyase gene cDNA partial sequence was 487 bp long, coding 159 amino acids (accession number: GU355673).
2. In the present study, we have amplified the PAL gene fragment using mRNA extracted from the fruit, cloned it in a T-vector, identified by sequencing and further constructed in the plant expression vectors. The analysis showed that the partial sequence, which were 99%, 96%, 87% and 80% homology to that of Pyrus communis, Eriobotrya japonica, Prunus persica, Rubus idaeus PAL cDNA of Genbank, respectively.
3. To introduce proper restriction sites for subsequent cloning into the plant expression vector, there was a site for MSEⅠ.
4. By doing the test of the hydrophobicity the result showed that the amino acids that constitute the Phenylalanine Ammonia-Lyase gene was lyophobic. And the amino acids included the 20 ordinary amino acids. The number between 3 to 11. And the Leu was the most which is 11% and the Pro was laest, 1%. In the 159 amino acids, 64 amino acids of them were lyophobic, which was 40%; 50 amino acids of them were water-loving, which was 34%; 21 amino acids were acidic, which was 12% and 24 amino acids were alkalescent, which was 14%.
1.根据苯丙氨酸解氨酶基因保守序列设计一对引物,以鸭梨果实cDNA为模板扩增得到478 bp的鸭梨苯丙氨酸解氨酶基因cDNA片段(GenBank登录号:GU355673),长度为478 bp,编码159个氨基酸残基。
2.利用RT-PCR技术从鸭梨果实中提取的mRNA中扩增出一个大小为478bp的木质素合成特异表达的PAL基因片段,将其克隆到pUCm-T Vector载体上获得了重组质粒,通过对该重组质粒进行了PCR扩增检测以及序列测序分析,证明我们插入到载体上的片段即是我们所扩增出来的DNA片段,对鸭梨苯丙氨酸解氨酶基因序列进行同源性比对,通过比对,其基因序列与GenBank中已登录的蔷薇科的西洋梨的同源性达99%;与枇杷、桃树、覆盆子的同源性分别为96%、87%和80%。从而证明我们所扩增出来的DNA片段即为PAL基因片段的一部分。
3.应用软件对所克隆的鸭梨苯丙氨酸解氨酶基因序列片段进行了酶切位点的检测,结果显示鸭梨苯丙氨酸解氨酶基因序列片段中含有大量的酶切位点,其中含有常用的限制性内切酶MSEⅠ的识别位点。
4.对所克隆的鸭梨苯丙氨酸解氨酶基因编码的氨基酸进行疏水性的检测,其所编码的氨基酸表现为疏水性。并对所克隆片段的氨基酸组成进行了分析,结果表明,鸭梨苯丙氨酸解氨酶基因推导蛋白中常见的二十种氨基酸均有涉及,数量在3~11之间,其中以Leu含量最高,为11%,以Pro含量为最低,仅占1%。在总共159个氨基酸中,共有疏水性氨基酸64个,占氨基酸总数的40%;亲水性氨基酸占50个,占总数的34%;酸性氨基酸21个,碱性氨基酸24个,分别占总数的12%和14%。
The economic values of the pears depend on the edible quality of the pear fruit. And the stone cell is one of the most factors. So the stone cell of the pear fruit takes the direct effect of the quality. The sediment of lignin comes into being the stone cell.The phenylalanine ammonia- lyase (PAL) which links primary and secondary metabolism by catalyzing the conversion of L-phenylalanine to cinnamic acid, is the first key enzyme in the pathway of plant secondary metabolism. The study which used Yali pear as starting material analyzes the sequence of Yali pear. The major results of this dissertation are as follows:
1. High purity RNA was extracted from Yali pear and used as a template for cloning the Phenylalanine Ammonia- Lyase gene cDNA by RT-PCR with specific primers under gradient temperature. Yali pear Phenylalanine Ammonia-Lyase gene cDNA partial sequence was 487 bp long, coding 159 amino acids (accession number: GU355673).
2. In the present study, we have amplified the PAL gene fragment using mRNA extracted from the fruit, cloned it in a T-vector, identified by sequencing and further constructed in the plant expression vectors. The analysis showed that the partial sequence, which were 99%, 96%, 87% and 80% homology to that of Pyrus communis, Eriobotrya japonica, Prunus persica, Rubus idaeus PAL cDNA of Genbank, respectively.
3. To introduce proper restriction sites for subsequent cloning into the plant expression vector, there was a site for MSEⅠ.
4. By doing the test of the hydrophobicity the result showed that the amino acids that constitute the Phenylalanine Ammonia-Lyase gene was lyophobic. And the amino acids included the 20 ordinary amino acids. The number between 3 to 11. And the Leu was the most which is 11% and the Pro was laest, 1%. In the 159 amino acids, 64 amino acids of them were lyophobic, which was 40%; 50 amino acids of them were water-loving, which was 34%; 21 amino acids were acidic, which was 12% and 24 amino acids were alkalescent, which was 14%.
引文
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