茶树CsMDHAR基因的克隆与非生物胁迫响应分析

Cloning and Response Analysis of the CsMDHAR Gene Under the Abiotic Stress in Camellia sinensis

基于茶树转录组数据,以龙井43茶树cDNA为模板,克隆得到开放阅读框(ORF)长度为1 305 bp,编码434个氨基酸的茶树单脱氢抗坏血酸还原酶基因,命名为CsMDHAR。蛋白序列特征和基因表达模式分析表明,CsMDHAR蛋白含有FAD结合功能域,属于FAD依赖的吡啶核苷酸-硫基氧化还原酶(Pyr-redox-2)家族。该蛋白有两个无序化区域,而且包含有32个磷酸化位点,理论相对分子量为47.21 kDa,pI为5.99,属于亲水性蛋白;CsMDHAR蛋白二级结构以α-螺旋和随机卷曲为主。通过PlantCARE和PLACE对启动子上游调控元件进行分析预测,结果显示,CsMDHAR基因启动子上游1 000 bp中含有多个与光、激素以及植物抗逆有关的顺式作用元件。利用荧光定量PCR方法检测龙井43和迎霜的CsMDHAR、CsAO和CsAPX在4种不同非生物胁迫下的表达水平,结果表明,在低温(4℃)胁迫下,两个茶树品种的CsMDHAR、CsAO和CsAPX的表达均受抑制,且品种间的差异较小;在高温(38℃)和干旱(200 g·L^-1 PEG)胁迫下龙井43中的CsMDHAR表达均上调,分别在8 h和2 h时达到最大值,为对照的1.49倍和1.85倍;盐(200 mmol·L^-1 NaCl)胁迫下,CsAO和CsAPX在两种茶树品种中的表达量变化趋势相似,但变化幅度不同,可能与品种间不同的抗逆性有关。

In this study,a MDHAR gene(CsMDHAR)was cloned from Camellia sinensis cv.‘Longjing 43’based on the transcriptome data of tea plant.Sequence analysis shows that the open reading frame length of CsMDHAR was 1 305 bp,encoding 434 amino acids with a molecular weight of approximately 47.21 kDa and the theoretical isoelectric point of 5.99.CsMDHAR was a hydrophilic protein,including two unordered regions and 32 phosphorylation sites.CsMDHAR belonged to Pyr-redox-2 super-family containing a highly conserved region called FAD domain,and mainly composed ofα-helix and random coil.PlantCARE and PLACE database prediction analysis suggest that there were many cis-elements related to light,hormones and stress resistance in the 1 000 bp upstream region of CsMDHAR gene.The expression profiles of CsMDHAR,CsAO and CsAPX in tea cv‘Longjing 43’and‘Yingshuang’under high temperature,low temperature,drought,and salt treatments were detected by qRT-PCR.The results indicate that the expression profiles of CsMDHAR,CsAO and CsAPX were suppressed under 4℃,and there were no significantly differences in‘Longjing 43’and‘Yingshuang’.However,the expression profiles of CsMDHAR gene were upregulated under 38℃or 200 g·L^-1 PEG treatments in‘Longjing 43’,with the highest 2.5 and 5 times of the control at 8 h and 2 h,respectively.In addition,the expression trends of CsAO and CsAPX in both cultivars were similar under NaCl(200 mmol·L^-1)treatment,but the variation ranges were different,which might be related to the different stress response in tea plant.