香蕉A、B基因组胆碱单加氧酶基因克隆及渗透胁迫下表达特性分析

Cloning and Expression Characteristics of Choline Monooxygenase Gene in Banana A and B Genomes under Osmotic Stress

胆碱单加氧酶(CMO)是高等植物体内甜菜碱合成过程中的关键酶,在植物抵御逆境生理过程中具有重要作用。在香蕉A、B参考基因组数据库中分别鉴定到1个CMO基因,对MaCMO与MbCMO基因组序列进行生物学信息分析,发现MbCMO可能由1个香蕉O-岩藻糖基转移酶家族蛋白编码基因与MbCMO基因串联形成。克隆湛江AA(ZJ;AA基因型)、巴西蕉(BX;AAA基因型)、广东大蕉(GD;AAB基因型)和金粉(JF;ABB基因型)的CMO基因编码序列并测序比对,在ZJ和BX中鉴定到基因CMO-A,GD和JF中鉴定到基因CMO-A、CMO-B1和CMO-B2,JF中鉴定到基因CMO-H。鉴定到的4种香蕉CMO基因中,有23个共同的高频密码子,密码子CUU和CCG分别是偏性最强和最弱的密码子。CMO-A蛋白与CMO-H蛋白的氨基酸数量最少,均为425个;CMO-B1蛋白的氨基酸数量最多,为470个,且二级结构最为复杂;CMO-B2蛋白分子量最大,为52.02 kDa;CMO-A分子量最小,为47.48 kDa;4种CMO均属于酸性蛋白,均不具有跨膜结构且均为亲水性蛋白;亚细胞定位预测显示4种CMO均定位在叶绿体中。在进化方面,植物CMO在进化时单、双子叶植物有明显的分支,香蕉CMO-A、CMO-H、CMO-B1、CMO-B2蛋白与其他单子叶植物CMO蛋白亲缘关系更近。RT-qPCR结果显示:4种香蕉根中CMO在渗透胁迫前期上调表达,A基因组纯合型ZJ与BX的CMO表达量高于A、B基因组杂合型GD与JF的CMO表达量;4种香蕉叶中CMO在渗透胁迫前期下调表达,其中A基因组纯合型ZJ与BX的CMO表达量在渗透胁迫后期出现上调表达,A、B基因组杂合型GD与JF的CMO表达量高峰出现在10 d,而后15 d时表达量再次下调,并显著低于ZJ与BX的CMO表达量。本研究揭示了香蕉A、B基因组间CMO基因的差异和不同基因型香蕉中CMO基因在渗透胁迫下的表达模式,为进一步研究香蕉A、B基因组CMO基因生物学功能,特别是来源于不同基因组的CMO基因与香蕉抗渗透胁迫能力间的关系奠定基础,为利用基因工程提高香蕉抗逆性提供参考依据。

Choline monooxygenase(CMO)is a key enzyme in the synthesis of betaine in higher plants and plays an im-role in the physiological process of plant resistance to stress.A CMO gene was identified in banana A and B reference genomes in banana gene database,and the biological information analysis of MaCMO and MbCMO genome sequences showed that MbCMO might be formed by a gene encoding a banana O-fucosyltransferase family protein and MbCMO gene in tandem.The CMO gene coding sequences of Zhanjiang AA(ZJ;AA genotype),Baxijiao(BX;AAA genotype),Guangdong Dajiao(GD;AAB genotype)and Jinfen(JF;ABB genotype)were cloned and compared,it was found that ZJ and BX only contained CMO-A,GD and JF both contained CMO-A,CMO-B1 and CMO-B2,and JF also contained CMO-H.Codon usage characteristics showed that there were 23 common high frequency codons among the four banana CMO gene,and the codons CUU and CCG were the most biased and the weakest codons,respectively.The physicochemical properties of CMO-A protein and CMO-H protein showed that the minimum number of amino acids was 425,the maximum number of amino acids was 470,and the secondary structure of CMO-B1 protein was the most complex.The molecular weight of CMO-B2 protein was 52.02 kDa,and the molecular weight of CMO-A protein was 47.48 kDa.All the four CMO proteins were acidic proteins,which did not have a transmembrane structure and were hydrophilic proteins.Subcellular localization prediction showed that all four CMO were localized in chloroplasts.In terms of evolution,plant CMO had obvious branches in monodicotyledonous plants during evolution,and the CMO-A,CMO-H,CMO-B1 and CMO-B2 proteins of banana were more closely related to other monocotyledonous plant CMO proteins.The results of RT-qPCR showed that CMO expression was up-regulated in the four kinds of banana roots in the early stage of osmotic stress,and the expression levels of ZJ and BX CMO in homozygous A genome were higher than those of GD and JF CMO in heterozygous A and B genomes.CMO expression in the four kinds of banana leaves was down-regulated at the early stage of osmotic stress.The expression levels of homozygous ZJ and BX CMO in A genome were up-regulated at the late stage of osmotic stress,while the expression levels of heterozygous GD and JF CMO in A and B genomes peaked at 10 days and then down-regulated again at 15 days later.It was significantly lower than that of ZJ and BX CMO.This study revealed the differences of CMO genes between the A and B genomes of bananas and the expression patterns of CMO genes in different genotypes of bananas under osmotic stress,which would lay a foundation for further research on the biological functions of CMO genes in the A and B genomes of bananas,especially the rela-tionship between CMO genes derived from different genomes and the ability of banana to resist osmotic stress.It would provide a reference for improving the stress resistance of banana by genetic engineering.