不结球白菜耐热相关基因BcPLDγ的克隆和表达分析

Clone and Expression Analysis of Heat Resistance Related Gene BcPLDγ from Non-heading Chinese Cabbage

以不结球白菜耐热自交系‘高华青’、‘苏州青’和热敏自交系‘矮脚黄’为试验材料,利用RACE技术克隆得到不结球白菜BcPLDγ基因,利用生物信息学方法对该基因进行信息学分析,并采用qRT-PCR技术对热胁迫处理条件下不结球白菜叶片BcPLDγ基因的表达模式进行了研究.结果显示,BcPLDγ基因的cDNA全长为2736bp,其中开放阅读框长度为1905bp,共编码634个氨基酸.该蛋白不存在信号肽序列,预测相对分子量为71.59kD,理论等电点为6.73.BcPLDγ蛋白的二级结构主要由α-螺旋、延伸直链、β-转角和无规则卷曲4种形式构成.该蛋白含有一个植物PLD特有的C2结构域和一个PLDc结构域.系统进化分析表明BcPLDγ蛋白与同属植物的进化关系相近,其中与甘蓝型油菜进化关系最近.热胁迫处理条件下,3份试验材料中BcPLDγ基因表达均显著上调,并于12h时达到峰值,且上调趋势与试验材料的耐热性呈正相关.

In this study,taking non-heading Chinese cabbage heat resistant inbred ＂ Gao Huaqing＂, ＂ Su Zhouqing＂ and susceptible inbred ＂ Ai Jiaohuang＂ as experimental materials,we obtained BcPLDγ gene using RACE technology.A series of bioinformatics software was used to speculate acid sequence and its nucleotide sequence of BcPLDγ.Under heat stress,gene expression patterns of BcPLDγ were investigated by using qRT-PCR in leaves.The results showed that the full length CDS of BcPLDγ gene was 2736 bp,containing a 1905 bp open reading frame to encode a total of 634 amino acid residues. Meanwhile,the BcPLDγ protein from non-heading Chinese cabbage had no signal peptide sequence.Prediction of the relative molecular mass of BcPLDγ was 71.59 KD with the theoretical isoelectric point of 6.73. The two-dimension structure of BcPLDγ protein mainly consisted of α-helixes,extended straight chain,β-angle and random coils.BcPLDγ protein contains a C2 domain and a PLDc domain which belong to plant PLD superfamily. Homology analysis showed that the homologous species should mainly belong to the cruciferae,the BcPLDγ from non-heading Chinese cabbage has a highly identity with that from Brassica napus.Under heat stress,gene expression of BcPLDγ in all experimental materials were up-regulated gradually and peaked at 12 h,and the up-regulated trend was positively correlated with the heat resistance of the experimental materials.