花生NADH脱氢酶1亚基10B-like基因克隆表达分析及载体构建

Cloning and Expression Vectors Construction of NADH Dehydrogenase 1 Beta Subcomplex Subunit 10B-like Gene from Peanut

NADH脱氢酶是线粒体氧化呼吸链上重要的复合物,对线粒体功能有至关重要的作用。本研究以花生品种"日花1号"为材料,利用RACE方法成功得到NADH脱氢酶1亚基10B-like基因(ND1-10B-like)的c DNA和DNA全长。基因开放阅读框为321 bp,编码106个氨基酸组成的肽链,预测的蛋白质分子量为12 570.29 Da,等电点为6.97。DNA序列包含一个内含子和两个外显子,共1 269 bp。检测了基因组织表达和胁迫表达情况,结果显示该基因在叶片中表达量最高,在青枯菌胁迫条件下0.5 h表达量即可升高到初始值的5.7倍,对生物胁迫反应非常迅速。并进一步构建了基因过表达载体p CAMBIA2300-OE-ND1和反义表达载体p CAMBIA2300-IN-ND1,并为进一步研究花生中该基因的功能及其在花生抗青枯病中的作用提供帮助。

NADH dehydrogenase is an important complex in the mitochondrial oxidative respiratory chain and plays a critical role in the mitochondrial function. In this study, the sequences of cDNA and DNA ofNADHI-10B- like （ND1-10B-like） were obtained successfully by RACE from A rachis hypogaea cultivar Ri Hua 1. The full length cDNA ofgene included a 321 bp open reading frame （ORF） which encodeds a peptide chain containing 106 amino acids. Primary structure analysis revealed that the molecular weight of the putative protein was 12 570.29 Da with a theoretical pI of 6.97. The full length DNA of gene was 1 269 bp with two exons and one intron. The gene expression and stress expression were detected. The results showed that the expression of this gene was highest in leaves. Under the condition of Ralstonia solanacearum, the expression amount of 0.5 h could increase to 5.7 times of initial value, indicating that it reacted very quickly to biotic stresses. The study also constructed overexpression vector pCAMBIA2300-OE-ND 1 study would be beneficial for the and antisense interference expression vector pCAMBIA2300-1N-ND1. This further study on the function of this gene in peanut and its role in peanutresistance to bacterial wilt.