香蕉MaGA20ox4和MaGA20ox5基因克隆、表达及亚细胞定位分析

段雅婕 陈经烨 陈晶晶

摘  要：以威廉斯香蕉（Musa spp. AAA group）‘8818及其突變体‘8818-1的假茎为试材，同源克隆MaGA20ox4和MaGA20ox5基因的gDNA及启动子序列，比较它们在基因组测序品种以及‘8818和‘8818-1中的序列差异。序列比对结果表明：MaGA20ox4和MaGA20ox5基因的gDNA相似性达93.82%，而二者的启动子序列相似性仅有40%，威廉斯品种与基因组测序品种间MaGA20ox4和MaGA20ox5基因无论gDNA还是启动子序列均存在较大差异，特别是启动子序列。威廉斯‘8818和‘8818-1间MaGA20ox4基因gDNA也存在一定的碱基序列差异，但启动子序列基本相同，MaGA20ox5结果同MaGA20ox4类似。启动子分析推测2个基因的启动子属于诱导性启动子，存在较多光响应和激素响应元件。进一步构建了MaGA20ox4和MaGA20ox5的亚细胞定位表达载体，定位结果显示，MaGA20ox4和MaGA20ox5蛋白均定位于细胞核上，可能定位于细胞质或细胞膜。在不同品种香蕉果实的不同发育时期qRT-PCR结果表明，MaGA20ox4和MaGA20ox5具有品种特异性，在威廉斯香蕉品种果实中可能不是主要的赤霉素合成调控基因。

关键词：香蕉;赤霉素20氧化酶;基因克隆;亚细胞定位

中图分类号：S668.1      文献标识码：A

Cloning， Expression and Subcellular Localization Analysis of MaGA20ox4 and MaGA20ox5 Gene from Banana

DUAN Yajie， CHEN Jingye， CHEN Jingjing^*

Institute of South Subtropical Crop Research Institute， Chinese Academy of Tropical Agricultural Sciences / Key Laboratory of Tropical Fruit Biology， Ministry of Agriculture and Rural Affairs / National Field Genebank for Tropical Fruit / Key Laboratory of Hainan Province for Postharvest Physiology and Technology of Tropical Horticultural Products， Zhanjiang， Guangdong 524091， China

Abstract： Using the false stem of Musa spp. AAA Group ‘8818 and its mutant ‘8818-1 as the materials， the gDNA and promoter sequences of MaGA20ox4 and MaGA20ox5 genes were cloned homogeneously， and the sequence differences in the genome-sequencing variety， ‘8818 and ‘8818-1 were compared. The results of sequence alignment showed that the gDNA similarity of MaGA20ox4 and MaGA20ox5 genes reached 93.82%， while the promoter sequence similarity of the two genes was only 40%. There were large differences in gDNA and promoter sequences of MaGA20ox4 and MaGA20ox5 genes between Williams varieties and genome-sequencing variety， especially the promoter sequence. The gDNA of MaGA20ox4 gene of Williams ‘8818 and ‘8818-1 also had difference in base sequence， but the promoter sequence was basically the same， and the result of MaGA20ox5 was similar to MaGA20ox4. Promoter analysis suggested that the promoters of the two genes belonged to inducible promoters， and there were more light response and hormone response elements. The subcellular localization vectors of MaGA20ox4 and MaGA20ox5 were further constructed. The localization results showed that MaGA20ox4 and MaGA20ox5 proteins were localized on the nucleus and might be localized on the cell membrane or cytoplasm. Quantitative RT-PCR results at different developmental stages of different banana fruits showed that MaGA20ox4 and MaGA20ox5 were species-specific， and may not be the main gene regulating gibberellin synthesis in Williams banana fruits.

Keywords： banana; GA20ox; gene clone; subcellular localization

DOI： 10.3969/j.issn.1000-2561.2021.07.001

赤霉素（GA）作为调控植物发育的重要激素，最明显的生理作用是促进伸长，但不增加节间^[1]。赤霉素氧化酶是调控赤霉素代谢的关键酶，其中GA-20氧化酶（GA20ox）和GA3氧化酶（GA3ox）能将GA₁₂和GA₅₃催化合成有活性的GA₁和GA₄;而GA2氧化酶（GA2ox）能通过β-羟基化作用将活性GA₁和GA₄催化形成无活性的GA₈和GA₃₄，这些关键基因的表达同时受到活性赤霉素的反馈和前馈调节^[1-3]。赤霉素氧化酶属于多基因家族，研究发现，水稻中存在8个GA20ox、2个GA3ox、11个GA2ox基因;拟南芥中存在5个GA20ox、4个GA3ox、8个GA2ox基因;大豆中存在8个GA20ox、6个GA3ox、10个GA2ox基因^[4]。……