本文主要研究内容
作者熊兴鹏（2019）在《植物PME基因家族的分析及白菜PME37c的功能鉴定》一文中研究指出：花粉发育是植物有性生殖的重要一环,花粉壁的正常发育对于花粉形态和功能的维持尤为重要。花粉内壁主要由果胶等组成,果胶的合成和修饰过程若有异常,则会直接影响内壁的发育,进而造成花粉萌发及花粉管生长等异常。果胶甲酯酶(pectinmethylesterase,PME)是果胶侧链的一种重要修饰酶,已有研究表明,在拟南芥和白菜中,有数个PME基因参与花粉内壁的形成。拟南芥中至少有15个PME参与花粉发育,白菜中也有20多个PME与花粉发育相关,但其中绝大部分PME基因的具体功能还不清楚。研究白菜基因功能的传统方法主要有诱变、反义干涉、RNA干涉、过表达等,但由于白菜在进化过程中经历了一次全基因组三倍化事件,导致其基因组内同源基因比较多,上述方法在实际应用过程中存在很多局限。新兴的CRISPR/Cas9基因编辑技术可以很好地解决这些问题,但白菜中尚未建立起成熟的CRISPR/Cas9体系。本文首先通过生物信息学的方法对白菜等九种植物的PME基因家族成员进行鉴定,再结合网上公布的转录组数据对这些PME基因进行表达分析;然后找出各个植物中花发育特异或优势表达的PME,对其进行序列比对和启动子顺式作用元件预测;继而以白菜(Brassica campestris L.subsp.chinensis Makino,syn.B.rapa subsp.chinensis)’矮脚黄’核不育(’Aijiaohuang’genic male sterility,ajhGMS)两用系’Bcajh97-01A/B’为材料,采用qRT-PCR技术进一步确认白菜PME家族成员的表达,特别是 BcPME37a(BraA09g051660.3C)、BcPME37b(BraA04g000830.3C)和 B)和BcPME37c(BraA07g025040.3C)三个基因在可育系花发育中的表达特点;随后,以’油青四九’菜心(B.campestris subsp.chinensis var.parachinensis cv.Youqing 49)为材料,采用 CRISPR/Cas9技术对三个PME基因进行基因编辑,再综合运用进行形态学、分子生物学、细胞学等方法分析BcPME37c在白菜花粉发育中的功能。取得的主要结果与结论如下:(1)在白菜等九种植物中共鉴定到了709个PME基因,每种植物的PME家族成员数量都很多。对PME家族成员进行表达分析,发现有相当大一部分PME基因参与花粉的发育。MEME程序分析结果表明这些花发育相关的PME在蛋白序列上有较多保守基序。进一步用PlantCARE工具分析其启动子,结果表明这些PME也有很多相同的顺式作用元件。这些结果说明花粉发育相关PME在不同的植物中极其保守。(2)根据白菜基因组数据库的参考序列设计引物,在’油青四九’菜心中克隆得到了BcPME37a、BcPME37b和BcPME37c的DNA序列和CDS。比对结果表明这三个基因相似度很高,且与拟南芥PME37(At3g62170)有很高的相似性。进一步通过qRT-PCR分析发现BcPME37a、BcPME37b和BcPME37c均在成熟花粉时期表达水平较高。授粉后数小时的雌蕊中,BcPME37a和BcPME37b仍有较高的表达,但是BcPME37c却几乎没有表达。启动子融合GUS转化拟南芥的实验进一步证实了BcPME37c在成熟花粉时期的花药中表达量最高。BcPME37a、BcPME37b和BcPME37c是拟南芥PME37在白菜中的同源基因,但它们三个的表达特点不尽相同,表明它们可能有功能分化。(3)对拟南芥CRISPR/Cas9载体进行改造,针对BcPME37a、BcPME37b和BcPME37c设计了三条sgRNA,并成功构建了 pBI121骨架的CRISPR/Cas9载体。对’油青四九’菜心的遗传转化得到了多个基因编辑的芽系,编辑效率可达到50%。不仅实现了BcPME37c单靶点的基因编辑,还实现了BcPME37b和BcPME37c两个靶点的同时编辑。对预测的潜在脱靶位点进行测序,发现基因编辑芽系中没有发生脱靶。bcpme37c自交留种,后代中检测到了不含T-DNA片段的植株。(4)对bcpme37c观察发现,相比于对照植株,突变体的营养生长过程并无明显差异,花器官发育正常,结籽率没有变化。进一步对花粉进行亚历山大染色,发现BcPME37c的敲除突变体的花粉吸胀后有约5%的花粉直径明显增大。扫描电镜观察结果显示突变体约5%的花粉畸形,畸形的比例与吸胀后直径增大的花粉的比例相当。透射电镜观察结果表明,突变体在双核花粉期和三核花粉期花粉内壁异常加厚。BcPME37c被敲除之后,花粉内壁中同聚半乳糖醛酸(Homogalacturonan,HG)的甲酯化程度很可能异常升高,同时花粉特异表达的其他PME可能上调表达,最终导致花粉内壁异常加厚,进而影响花粉的吸胀和花粉的形态。
Abstract
hua fen fa yo shi zhi wu you xing sheng shi de chong yao yi huan ,hua fen bi de zheng chang fa yo dui yu hua fen xing tai he gong neng de wei chi you wei chong yao 。hua fen nei bi zhu yao you guo jiao deng zu cheng ,guo jiao de ge cheng he xiu shi guo cheng re you yi chang ,ze hui zhi jie ying xiang nei bi de fa yo ,jin er zao cheng hua fen meng fa ji hua fen guan sheng chang deng yi chang 。guo jiao jia zhi mei (pectinmethylesterase,PME)shi guo jiao ce lian de yi chong chong yao xiu shi mei ,yi you yan jiu biao ming ,zai ni na gai he bai cai zhong ,you shu ge PMEji yin can yu hua fen nei bi de xing cheng 。ni na gai zhong zhi shao you 15ge PMEcan yu hua fen fa yo ,bai cai zhong ye you 20duo ge PMEyu hua fen fa yo xiang guan ,dan ji zhong jue da bu fen PMEji yin de ju ti gong neng hai bu qing chu 。yan jiu bai cai ji yin gong neng de chuan tong fang fa zhu yao you you bian 、fan yi gan she 、RNAgan she 、guo biao da deng ,dan you yu bai cai zai jin hua guo cheng zhong jing li le yi ci quan ji yin zu san bei hua shi jian ,dao zhi ji ji yin zu nei tong yuan ji yin bi jiao duo ,shang shu fang fa zai shi ji ying yong guo cheng zhong cun zai hen duo ju xian 。xin xing de CRISPR/Cas9ji yin bian ji ji shu ke yi hen hao de jie jue zhe xie wen ti ,dan bai cai zhong shang wei jian li qi cheng shou de CRISPR/Cas9ti ji 。ben wen shou xian tong guo sheng wu xin xi xue de fang fa dui bai cai deng jiu chong zhi wu de PMEji yin jia zu cheng yuan jin hang jian ding ,zai jie ge wang shang gong bu de zhuai lu zu shu ju dui zhe xie PMEji yin jin hang biao da fen xi ;ran hou zhao chu ge ge zhi wu zhong hua fa yo te yi huo you shi biao da de PME,dui ji jin hang xu lie bi dui he qi dong zi shun shi zuo yong yuan jian yu ce ;ji er yi bai cai (Brassica campestris L.subsp.chinensis Makino,syn.B.rapa subsp.chinensis)’ai jiao huang ’he bu yo (’Aijiaohuang’genic male sterility,ajhGMS)liang yong ji ’Bcajh97-01A/B’wei cai liao ,cai yong qRT-PCRji shu jin yi bu que ren bai cai PMEjia zu cheng yuan de biao da ,te bie shi BcPME37a(BraA09g051660.3C)、BcPME37b(BraA04g000830.3C)he B)he BcPME37c(BraA07g025040.3C)san ge ji yin zai ke yo ji hua fa yo zhong de biao da te dian ;sui hou ,yi ’you qing si jiu ’cai xin (B.campestris subsp.chinensis var.parachinensis cv.Youqing 49)wei cai liao ,cai yong CRISPR/Cas9ji shu dui san ge PMEji yin jin hang ji yin bian ji ,zai zeng ge yun yong jin hang xing tai xue 、fen zi sheng wu xue 、xi bao xue deng fang fa fen xi BcPME37czai bai cai hua fen fa yo zhong de gong neng 。qu de de zhu yao jie guo yu jie lun ru xia :(1)zai bai cai deng jiu chong zhi wu zhong gong jian ding dao le 709ge PMEji yin ,mei chong zhi wu de PMEjia zu cheng yuan shu liang dou hen duo 。dui PMEjia zu cheng yuan jin hang biao da fen xi ,fa xian you xiang dang da yi bu fen PMEji yin can yu hua fen de fa yo 。MEMEcheng xu fen xi jie guo biao ming zhe xie hua fa yo xiang guan de PMEzai dan bai xu lie shang you jiao duo bao shou ji xu 。jin yi bu yong PlantCAREgong ju fen xi ji qi dong zi ,jie guo biao ming zhe xie PMEye you hen duo xiang tong de shun shi zuo yong yuan jian 。zhe xie jie guo shui ming hua fen fa yo xiang guan PMEzai bu tong de zhi wu zhong ji ji bao shou 。(2)gen ju bai cai ji yin zu shu ju ku de can kao xu lie she ji yin wu ,zai ’you qing si jiu ’cai xin zhong ke long de dao le BcPME37a、BcPME37bhe BcPME37cde DNAxu lie he CDS。bi dui jie guo biao ming zhe san ge ji yin xiang shi du hen gao ,ju yu ni na gai PME37(At3g62170)you hen gao de xiang shi xing 。jin yi bu tong guo qRT-PCRfen xi fa xian BcPME37a、BcPME37bhe BcPME37cjun zai cheng shou hua fen shi ji biao da shui ping jiao gao 。shou fen hou shu xiao shi de ci rui zhong ,BcPME37ahe BcPME37breng you jiao gao de biao da ,dan shi BcPME37cque ji hu mei you biao da 。qi dong zi rong ge GUSzhuai hua ni na gai de shi yan jin yi bu zheng shi le BcPME37czai cheng shou hua fen shi ji de hua yao zhong biao da liang zui gao 。BcPME37a、BcPME37bhe BcPME37cshi ni na gai PME37zai bai cai zhong de tong yuan ji yin ,dan ta men san ge de biao da te dian bu jin xiang tong ,biao ming ta men ke neng you gong neng fen hua 。(3)dui ni na gai CRISPR/Cas9zai ti jin hang gai zao ,zhen dui BcPME37a、BcPME37bhe BcPME37cshe ji le san tiao sgRNA,bing cheng gong gou jian le pBI121gu jia de CRISPR/Cas9zai ti 。dui ’you qing si jiu ’cai xin de wei chuan zhuai hua de dao le duo ge ji yin bian ji de ya ji ,bian ji xiao lv ke da dao 50%。bu jin shi xian le BcPME37cchan ba dian de ji yin bian ji ,hai shi xian le BcPME37bhe BcPME37cliang ge ba dian de tong shi bian ji 。dui yu ce de qian zai tuo ba wei dian jin hang ce xu ,fa xian ji yin bian ji ya ji zhong mei you fa sheng tuo ba 。bcpme37czi jiao liu chong ,hou dai zhong jian ce dao le bu han T-DNApian duan de zhi zhu 。(4)dui bcpme37cguan cha fa xian ,xiang bi yu dui zhao zhi zhu ,tu bian ti de ying yang sheng chang guo cheng bing mo ming xian cha yi ,hua qi guan fa yo zheng chang ,jie zi lv mei you bian hua 。jin yi bu dui hua fen jin hang ya li shan da ran se ,fa xian BcPME37cde qiao chu tu bian ti de hua fen xi zhang hou you yao 5%de hua fen zhi jing ming xian zeng da 。sao miao dian jing guan cha jie guo xian shi tu bian ti yao 5%de hua fen ji xing ,ji xing de bi li yu xi zhang hou zhi jing zeng da de hua fen de bi li xiang dang 。tou she dian jing guan cha jie guo biao ming ,tu bian ti zai shuang he hua fen ji he san he hua fen ji hua fen nei bi yi chang jia hou 。BcPME37cbei qiao chu zhi hou ,hua fen nei bi zhong tong ju ban ru tang quan suan (Homogalacturonan,HG)de jia zhi hua cheng du hen ke neng yi chang sheng gao ,tong shi hua fen te yi biao da de ji ta PMEke neng shang diao biao da ,zui zhong dao zhi hua fen nei bi yi chang jia hou ,jin er ying xiang hua fen de xi zhang he hua fen de xing tai 。
论文参考文献
论文详细介绍
论文作者分别是来自浙江大学的熊兴鹏，发表于刊物浙江大学2019-09-18论文，是一篇关于白菜论文,花粉发育论文,花粉内壁论文,基因家族论文,浙江大学2019-09-18论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自浙江大学2019-09-18论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：白菜论文; 花粉发育论文; 花粉内壁论文; 基因家族论文; 浙江大学2019-09-18论文;