本文主要研究内容
作者朱守鸿（2019）在《棉花纤维发育相关基因GhCLASP2和GhAlaRP功能研究》一文中研究指出：目的:棉花是世界上最重要的天然纤维和纺织工业材料来源。棉纤维是由棉花胚珠表皮细胞分化、发育而成。在棉纤维发育的过程中,有许多基因参与调控。植物细胞骨架的主要组分之一-微管在棉纤维伸长发育和次生壁加厚中也发挥着重要的功能。CLASP蛋白(CLIP-Associated Protein)能够与微管结合,参与调节微管结构与功能。已有研究表明CLASP基因与根和下胚轴细胞的伸长、信号转导及叶表皮毛分支等相关。棉纤维是棉花种子的表皮毛,由于结构和遗传上的相似性,棉纤维细胞与拟南芥表皮毛发育可能具有相似的调控机制。GhAlaRP(Ala-Rich-Protein)在棉花纤维中特异表达,在棉花纤维发育的伸长期(6 DPA)表达水平最高,推测该基因可能与棉纤维伸长发育相关。鉴于GhCLASP和GhAlaRP基因在纤维发育中可能存在重要的调控作用。本研究拟从棉纤维中克隆CLASP和AlaRP基因,解析其在棉纤维发育过程中的调控功能与分子机制,为利用基因工程改良棉纤维品质奠定基础。方法:对克隆获得的GhCLASP2和GhAlaRP基因序列进行生物信息学分析;构建GhCLASP2和GhAlaRP基因的植物亚细胞定位载体、过表达载体和RNAi(RNA interference)载体,分别浸染烟草(N.benthamiana)、拟南芥(Col-0和clasp-1)和棉花(YZ-1);对获得的转基因植物进行鉴定及表型观察,阐明GhCLASP2和GhAlaRP的功能。采用酵母双杂交和BIFC(Bimolecular fluorescence complementation),鉴定与GhAlaRP互作的蛋白,解析其在棉花纤维发育中的蛋白调控网络。构建CRISPR/Cas9-GhAlaRP基因编辑载体,浸染棉花(YZ-1),对获得转基因棉花进行鉴定,为研究GhAlaRP的功能提供突变体材料。结果与结论:(1)从陆地棉纤维中克隆获得了一个与AtCLASP序列同源的GhCLASP2基因。生物信息学分析表明,GhCLASP2含有2个HEAT重复结构域和2个CLASP-N结构域,属于CLASP家族I亚家族。qRT-PCR分析表明GhCLASP2在棉花纤维中优势表达,尤其在棉花纤维发育的次生壁加厚期(27 DPA和30 DPA)表达水平最高;表明GhCLASP2参与棉花纤维发育调控,可能和棉花纤维品质形成相关。亚细胞定位结果表明,GhCLASP2定位在细胞中的微管上。GhCLASP2基因异位表达拟南芥叶片表皮毛分支数目增加,并能恢复拟南芥clasp-1突变体表皮毛、下胚轴和根系的表型。与对照YZ-1株系相比,过表达GhCLASP2转基因棉花株系纤维强度显著增强,这可能与微管蛋白(TUA和TUB)、纤维素合成酶(CESA)和延伸蛋白(EXP)基因的表达水平变化相关;GhCLASP2-RNAi转基因株系表现出植株矮小、节间缩短、柱头异常、雄蕊无花粉的表型。这些现象与拟南芥突变体clasp-1的表型相似。以上结果表明,棉花GhCLASP2和拟南芥AtCLASP可能具有相似的功能,通过调控微管来调节棉花的生长发育。(2)GhAlaRP是一个含有2个跨膜结构域的蛋白,与可可树AlaRP同源关系最近。亚细胞定位表明,GhAlaRP定位在细胞质膜、细胞核和内质网上,表明GhAlaRP参与棉花纤维细胞中的物质运输。酵母双杂交和BIFC结果表明,GhAlaRP分别与膜联相关蛋白GhAnnexin(Gh_D11G2184)和延伸蛋白GhEXPA(Gh_A10G2323)相互作用。抑制GhAlaRP表达的转基因棉花纤维长度变短,同时GhAnnexin和GhEXPA表达水平亦显著降低。上述结果表明,GhAlaRP参与调控棉花纤维伸长发育,干扰该基因表达其纤维长度显著下降,GhAlaRP通过与GhAnnexin和GhEXPA蛋白相互作用,形成一个调控网络来调节棉花纤维的发育,在此过程中可能还有其他蛋白质的参与。此外,对CRISPR/Cas9基因编辑获得的棉花alarp突变体纤维进行观察,突变体纤维长度较对照YZ-1纤维显著变短。与GhAlaRP-RNAi植株纤维的表型一致,这些结果进一步表明GhAlaRP参与调控棉花纤维发育的伸长。
Abstract
mu de :mian hua shi shi jie shang zui chong yao de tian ran qian wei he fang zhi gong ye cai liao lai yuan 。mian qian wei shi you mian hua pei zhu biao pi xi bao fen hua 、fa yo er cheng 。zai mian qian wei fa yo de guo cheng zhong ,you hu duo ji yin can yu diao kong 。zhi wu xi bao gu jia de zhu yao zu fen zhi yi -wei guan zai mian qian wei shen chang fa yo he ci sheng bi jia hou zhong ye fa hui zhao chong yao de gong neng 。CLASPdan bai (CLIP-Associated Protein)neng gou yu wei guan jie ge ,can yu diao jie wei guan jie gou yu gong neng 。yi you yan jiu biao ming CLASPji yin yu gen he xia pei zhou xi bao de shen chang 、xin hao zhuai dao ji xie biao pi mao fen zhi deng xiang guan 。mian qian wei shi mian hua chong zi de biao pi mao ,you yu jie gou he wei chuan shang de xiang shi xing ,mian qian wei xi bao yu ni na gai biao pi mao fa yo ke neng ju you xiang shi de diao kong ji zhi 。GhAlaRP(Ala-Rich-Protein)zai mian hua qian wei zhong te yi biao da ,zai mian hua qian wei fa yo de shen chang ji (6 DPA)biao da shui ping zui gao ,tui ce gai ji yin ke neng yu mian qian wei shen chang fa yo xiang guan 。jian yu GhCLASPhe GhAlaRPji yin zai qian wei fa yo zhong ke neng cun zai chong yao de diao kong zuo yong 。ben yan jiu ni cong mian qian wei zhong ke long CLASPhe AlaRPji yin ,jie xi ji zai mian qian wei fa yo guo cheng zhong de diao kong gong neng yu fen zi ji zhi ,wei li yong ji yin gong cheng gai liang mian qian wei pin zhi dian ding ji chu 。fang fa :dui ke long huo de de GhCLASP2he GhAlaRPji yin xu lie jin hang sheng wu xin xi xue fen xi ;gou jian GhCLASP2he GhAlaRPji yin de zhi wu ya xi bao ding wei zai ti 、guo biao da zai ti he RNAi(RNA interference)zai ti ,fen bie jin ran yan cao (N.benthamiana)、ni na gai (Col-0he clasp-1)he mian hua (YZ-1);dui huo de de zhuai ji yin zhi wu jin hang jian ding ji biao xing guan cha ,chan ming GhCLASP2he GhAlaRPde gong neng 。cai yong jiao mu shuang za jiao he BIFC(Bimolecular fluorescence complementation),jian ding yu GhAlaRPhu zuo de dan bai ,jie xi ji zai mian hua qian wei fa yo zhong de dan bai diao kong wang lao 。gou jian CRISPR/Cas9-GhAlaRPji yin bian ji zai ti ,jin ran mian hua (YZ-1),dui huo de zhuai ji yin mian hua jin hang jian ding ,wei yan jiu GhAlaRPde gong neng di gong tu bian ti cai liao 。jie guo yu jie lun :(1)cong liu de mian qian wei zhong ke long huo de le yi ge yu AtCLASPxu lie tong yuan de GhCLASP2ji yin 。sheng wu xin xi xue fen xi biao ming ,GhCLASP2han you 2ge HEATchong fu jie gou yu he 2ge CLASP-Njie gou yu ,shu yu CLASPjia zu Iya jia zu 。qRT-PCRfen xi biao ming GhCLASP2zai mian hua qian wei zhong you shi biao da ,you ji zai mian hua qian wei fa yo de ci sheng bi jia hou ji (27 DPAhe 30 DPA)biao da shui ping zui gao ;biao ming GhCLASP2can yu mian hua qian wei fa yo diao kong ,ke neng he mian hua qian wei pin zhi xing cheng xiang guan 。ya xi bao ding wei jie guo biao ming ,GhCLASP2ding wei zai xi bao zhong de wei guan shang 。GhCLASP2ji yin yi wei biao da ni na gai xie pian biao pi mao fen zhi shu mu zeng jia ,bing neng hui fu ni na gai clasp-1tu bian ti biao pi mao 、xia pei zhou he gen ji de biao xing 。yu dui zhao YZ-1zhu ji xiang bi ,guo biao da GhCLASP2zhuai ji yin mian hua zhu ji qian wei jiang du xian zhe zeng jiang ,zhe ke neng yu wei guan dan bai (TUAhe TUB)、qian wei su ge cheng mei (CESA)he yan shen dan bai (EXP)ji yin de biao da shui ping bian hua xiang guan ;GhCLASP2-RNAizhuai ji yin zhu ji biao xian chu zhi zhu ai xiao 、jie jian su duan 、zhu tou yi chang 、xiong rui mo hua fen de biao xing 。zhe xie xian xiang yu ni na gai tu bian ti clasp-1de biao xing xiang shi 。yi shang jie guo biao ming ,mian hua GhCLASP2he ni na gai AtCLASPke neng ju you xiang shi de gong neng ,tong guo diao kong wei guan lai diao jie mian hua de sheng chang fa yo 。(2)GhAlaRPshi yi ge han you 2ge kua mo jie gou yu de dan bai ,yu ke ke shu AlaRPtong yuan guan ji zui jin 。ya xi bao ding wei biao ming ,GhAlaRPding wei zai xi bao zhi mo 、xi bao he he nei zhi wang shang ,biao ming GhAlaRPcan yu mian hua qian wei xi bao zhong de wu zhi yun shu 。jiao mu shuang za jiao he BIFCjie guo biao ming ,GhAlaRPfen bie yu mo lian xiang guan dan bai GhAnnexin(Gh_D11G2184)he yan shen dan bai GhEXPA(Gh_A10G2323)xiang hu zuo yong 。yi zhi GhAlaRPbiao da de zhuai ji yin mian hua qian wei chang du bian duan ,tong shi GhAnnexinhe GhEXPAbiao da shui ping yi xian zhe jiang di 。shang shu jie guo biao ming ,GhAlaRPcan yu diao kong mian hua qian wei shen chang fa yo ,gan rao gai ji yin biao da ji qian wei chang du xian zhe xia jiang ,GhAlaRPtong guo yu GhAnnexinhe GhEXPAdan bai xiang hu zuo yong ,xing cheng yi ge diao kong wang lao lai diao jie mian hua qian wei de fa yo ,zai ci guo cheng zhong ke neng hai you ji ta dan bai zhi de can yu 。ci wai ,dui CRISPR/Cas9ji yin bian ji huo de de mian hua alarptu bian ti qian wei jin hang guan cha ,tu bian ti qian wei chang du jiao dui zhao YZ-1qian wei xian zhe bian duan 。yu GhAlaRP-RNAizhi zhu qian wei de biao xing yi zhi ,zhe xie jie guo jin yi bu biao ming GhAlaRPcan yu diao kong mian hua qian wei fa yo de shen chang 。
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论文作者分别是来自石河子大学的朱守鸿，发表于刊物石河子大学2019-09-26论文，是一篇关于棉花纤维论文,伸长论文,基因编辑论文,石河子大学2019-09-26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，资料来自石河子大学2019-09-26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：棉花纤维论文; 伸长论文; 基因编辑论文; 石河子大学2019-09-26论文;