番木瓜果实软化相关CpEXPA2基因的克隆与表达分析  

Cloning and expression analysis of CpEXPA2 gene related to softening of papaya fruit

作　　者：赵湾湾 冯力 胡绍彬 代亚兰 李春侠 赵秋月 郑小华 吴迪 王平 申艳红[2] ZHAO Wanwan;FENG Li;HU Shaobin;DAI Yalan;LI Chunxia;ZHAO Qiuyue;ZHENG Xiao- hua;WU Di;WANG Ping;SHEN Yanhong(Institute of Genetics and Breeding in Horticultural Plants,College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;College of Horticultural Science and Technology,Hebei Normal University of Science and Technology,Qinhuangdao 066604,Hebei,China)

机构地区：[1]福建农林大学园艺学院园艺植物遗传育种研究所,福州350002 [2]河北科技师范学院园艺科技学院,河北秦皇岛066604

出　　处：《果树学报》2018年第7期785-793,共9页Journal of Fruit Science

基　　金：福建农林大学科技发展基金项目(KFA17355A)

摘　　要：【目的】克隆一个与番木瓜果实软化相关的扩展蛋白基因,分析其功能,明确其在不同组织器官和不同成熟时期果实的表达模式。【方法】基于对番木瓜果实转录组学的研究,筛选并克隆了一个与果实软化相关的扩展蛋白基因Cp EXPA2。采用同源序列对比和系统进化树分析对该基因进行序列分析;运用生物信息学的方法对该基因编码的蛋白进行结构分析;使用RT-q PCR方法分析Cp EXPA2基因在不同组织器官、不同成熟时期果实中的表达量;采用GY-4硬度计测定不同成熟时期番木瓜果实的硬度。【结果】Cp EXPA2基因的开放阅读框(ORF)为780 bp(Gene Bank登录号为MF662209),编码259个氨基酸。该基因编码的氨基酸序列具有典型的扩展蛋白保守结构:N端富含8个半胱氨酸、C端富含4个色氨酸和中间1个组氨酸功能域(His-Phe-Asp,HFD)。同源序列比对分析发现该扩展蛋白与山木瓜(ABF48653.1)、番茄(AAC64201.1)、草莓(AAF21101.1)、拟南芥(AAB38073.1)等扩展蛋白氨基酸序列有较高的同源性,分别为66.15%、64.86%、66.80%、65.00%。进化树分析表明,Cp EXPA2蛋白与拟南芥At EXPA6(U30480)、At EXPA16(NM_115407)关系最近。Cp EXPA2在不同组织器官中的表达量依次为成熟果实>叶>花>茎>根;在不同发育时期的果实中,Cp EXPA2在果皮的表达量相对高于果肉,且随着果实成熟软化程度提高,其表达量也相应提高。番木瓜果实的硬度随着果实的成熟呈逐渐下降趋势,在破色期开始硬度急剧下降,果实迅速软化。【结论】Cp EXPA2基因的扩展蛋白家族高度保守,且该基因编码的蛋白与山木瓜、番茄、草莓、拟南芥等扩展蛋白的氨基酸序列有较高的同源性。系统进化树分析发现番木瓜Cp EXPA2与拟南芥At EXPA6和At EXPA16亲缘关系较近。Cp EXPA2受乙烯诱导,且与果实发育进程有关,因此推测该基因可能在番木瓜果实成熟软化过程中发挥着重要的作用。【Objective】Expansins are a special group of plant cell wall proteins with no catalytic activity and are associated with several processes during plant growth and development. In particular, expansins play a role in fruit softening. Since a fruit-specific expansin gene Le EXPA1 was found from tomato fruit, ripening-related expansins had been identified in tomato, strawberry, banana, and a range of other fruit species. Papaya（Carica papaya L.） is an important fruit crop with high economic and nutritional value.‘Da Qing No. 10＇is a red-fleshed papaya. The red-fleshed papaya softens faster than the yellow-fleshed papaya and has a shorter shelf life. However, some consumers prefer red-fleshed papaya. Therefore, it is of great importance to explore the roles of expansins in fruit softening of papaya. An expansin gene was cloned and its function and expression patterns in different tissues and different fruit development and ripening stages were analyzed in this study.【Methods】Nineteen expansin candidate genes were selected according to gene annotation.in the papaya transcriptome data.Then the heat map analysis was carried out using the relative expression of gene FPKM value and a gene related to the softening of papaya fruit was found. Total RNA was extracted from papaya pulp using General RNA the kit R1051 of Guangzhou Dongsheng Biotechnology Co. Ltd. Referring to BD Biosciences Clontech, Super SMARTTMPCR c DNA Synthesis Kit manual, double-stranded c DNA was synthesized by using papaya RNA（200 ng） as a template. And amplification was carried out as follows： initial denaturation at 94 ℃for 3 min, followed by 35 cycles of 94 ℃（30 s）, 56.4 ℃（30 s）, and 72 ℃（2 min）, and then a final extension at 72 ℃ for 7 min. The PCR products were gel purified. The purified products were cloned into a p MD18-T vector（Ta Ka Ra） and transformed into E. coli DH5α competent cells, then the bacteria were spread onto LB Plates and incubated at 37 ℃ overnight. The positive clones were selected

关 键 词：番木瓜 扩展蛋白 表达模式 果实成熟软化 

分 类 号：S667.9[农业科学—果树学]