柚CmWOX2基因克隆及在胚发育过程中的表达分析  

Cloning and expressions analysis of CmWOX2 from Citrus maxima(Burm.)Merr.during embryo development

作　　者：靳三鹏 蒙志鑫 胡威 匡柳青 文婷 刘勇[1] 杨莉[1] JIN Sanpeng;MENG Zhixin;HUWei;KUANG Liuqing;WEN Ting;LIU Yong;YANG Li(School of Agriculture Sciences,Jiangxi Agricultural University,Nanchang 330000,Jiangxi,China)

机构地区：[1]江西农业大学农学院,南昌330000

出　　处：《果树学报》2022年第8期1337-1345,共9页Journal of Fruit Science

基　　金：国家自然科学基金项目(31960582,31860539);江西省柑橘产业技术体系项目(JXARS-07-栽培岗位);江西省自然科学基金项目(20202BAB215006)。

摘　　要：【目的】克隆马家柚[Citrus maxima(L.)Osbeck‘Majiayou’]WOX2基因,探讨CmWOX2基因在柚种子发育过程中的调控作用。【方法】使用PCR技术克隆柚CmWOX2基因,对其进行生物信息学分析,通过qRT-PCR技术比较、分析经花粉辐射处理后授粉获得的退化种子和正常果实中饱满种子中的CmWOX2基因在不同发育时期的表达情况。【结果】CmWOX2基因cDNA序列长度为1177 bp,序列同源性分析结果表明,柚CmWOX2基因与其他物种WOX2基因在5’homeobox区域具有高度相似性,在3’WUS-box区域有部分差异。实时荧光定量qRT-PCR结果表明,CmWOX2基因在马家柚的幼苗根、茎、叶中相对表达量较低,在种子中的相对表达量较高;CmWOX2基因在未受精的子房中也出现表达,受精后在马家柚胚的发育过程中(授粉后0~10周)相对表达量持续增加,之后随种子成熟,相对表达量逐渐下降;无核果实中的退化种子CmWOX2基因的相对表达量明显低于正常果实中的饱满种子。【结论】CmWOX2基因在马家柚胚胎的形成发育过程中发挥着至关重要的作用,并且通过花粉辐射获得的无核果实中CmWOX2基因相对表达量下降与胚发育异常密切相关。【Objective】The development of plant embryo is an extremely important part in the process of individual development.The morphogenesis of plant embryo is jointly determined by the MAPK(MAP KINASE)/GRD(GROUNDED)pathway and the WOX(WUSCHEL RELATED-HOMEBOX)transcription factor family members.The WOX transcription factor family members are differentially expressed in apical cells and basal cells,leading to the differentiation of different cells.WOX2 plays an important role in the early development of embryonic cells and the morphogenesis of stem apical meristems.This study aimed to explore the regulatory role of the CmWOX2 in seed development of Citrus maxima(Burm.)Merr.【Methods】The materials of this experiment were Majiayou trees from Shangrao,Jiangxi Province.The unirradiated and 60Co-γirradiated sour pomelo anthers were used for artificial pollination.The ovarys and seeds from the fruits were collected before and after pollination.The seeds derived from the pollination with the unirradiated pollens were sown in test tubes containing MS basic medium,and the roots,stems and leaves of the young plants were collected after 30 d culture(20 d dark culture,10 d light culture,culture temperature 26±1℃).The WOX2 gene sequence of the pomelo was obtained through search and comparison,and the full-length sequence of the CmWOX2 gene was obtained through amplification by designing specific primers.The PCR products were recovered and purified,and then were connected to the PMD18-T vector,and the positive clones were obtained through the blue-white screening for further sequencing analysis.DNAMAN,ExPASy ProtParam tool,TMHMM 2.0,SignalP 4.1 software were used for bioinformatics analysis.The expression of the CmWOX2 gene in degraded seeds obtained by pollination after pollen radiation treatment and full seeds in normal fruits were compared and analyzed by the qRT-PCR technology at different developmental stages.WPS2020 was used to sort and graph the data,SPSS 26.0 was used for significance analysis,the Duncan’s method(p

关 键 词：马家柚 WOX2基因 胚发育 表达分析 

分 类 号：S666.3[农业科学—果树学]