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作者王展超（2019）在《Pu-miR172d调控大青杨气孔发育响应干旱胁迫的机理研究》一文中研究指出：气孔是植物表皮由两个保卫细胞围绕形成的小孔,在调节植物光合固碳和水分散失的平衡方面影响颇大。气孔的发生是一种复杂的生物学过程,受多种基因所调控。气孔的发育不但可以影响植物的生长,也关系到植物应对环境胁迫的能力。目前,已有一些报道发现miRNA可以参与到气孔的发生过程,但其分子作用机制仍然不是很清晰。本研究旨在初步阐明大青杨miR172调控气孔发育响应干旱胁迫的分子机理。利用生物化学、分子生物学和基因工程等手段对大青杨Pu-miR172d及其靶基因PuGTL1的分子功能进行了分析鉴定。(1)小RNA转录组数据中靶基因预测结果发现Trihelix基因家族成员PuGTL1是Pu-miR172d的靶基因。利用5’RLM-RACE技术及农杆菌瞬时转化实验验证了 PuGTL1确实为Pu-miR172d的靶基因。首次在大青杨中发现了Pu-miR 72可以结合Trihelix家族的转录因子;(2)利用生物信息学方法和qRT-PCR技术对毛果杨Trihelix转录因子家族进行初步的分类及功能鉴定。Trihelix家族由56个成员构成,可分为五个亚族。其中,大多数基因都可以响应渗透胁迫。exImage分析结果表明PuGTL1主要在幼叶中表达。QRT-PCR结果显示PuGTL1下调表达在一定程度上可以提高植物抗逆性;(3)QRT-PCR结果显示Pu-miRI72d在第1-4片幼叶中表达量较高,并且其可以响应渗透胁迫。克隆大青杨Pu-miR172d启动子序列,构建Pu-miR172dpro::GUS表达载体。GUS染色后发现Pu-miR172d主要在叶片气孔中表达,暗示着Pu-miR172d可能参与早期的气孔运动或发育;(4)克隆全长大青杨Pu-miR172d序列,构建Pu-miR172d过表达和抑制表达载体。组培苗PEG6000渗透胁迫后发现,Pu-miR172d过表达转基因大青杨与WT相比,长势良好且生根率较高,而Pu-miR172d抑制表达转基因大青杨与WT差别不大。DAB、NBT和Evans bule组织化学染色及SOD、POD、MDA、电导率和叶绿素含量等生理指标测定结果显示Pu-miR172d过表达植株的抗逆性较强,说明Pu-miR172d具有一定的抗渗透胁迫能力;(5)Pu-miR172d过表达大青杨植株变矮,气孔密度显著减少,且净光合速率、气孔导度和蒸腾速率均低于WT,而瞬时水分利用率高于WT。Pu-miR172d过表达植株在干旱胁迫下叶片含水量更高,且干旱复水后,Pu-miR172d过表达植株可继续正常生长,而WT和Pu-miR172d抑制表达植株生长受到严重抑制。首次发现了 Pu-miR172d可以影响气孔发育,其可以通过减少气孔密度,降低蒸腾速率,提高瞬时水分利用率,从而提高植物的抗旱能力;(6)WT和Pu-miR172d过表达大青杨干旱处理前后的转录组数据中,有大量的与光合作用、失水胁迫和渗透胁迫响应的差异基因。干旱胁迫后,Pu-miR172d又促进了一些抗旱基因表达。QRT-PCR结果显示,PuGTL1在Pu-miR172d过表达植株中的转录水平明显下降,进一步说明了PuGTL 是Pu-miR72d的靶基因。但PuGTL1具有组织表达特异性,只在自上而下的第1-5片叶差异表达;(7)大青杨PuGTL1-SRDX抑制表达转基因大青杨土培苗与Pu-miR172d过表达植株在表型观察和生理指标测定结果方面基本一致,PuGTL的抑制表达也可使气孔密度减少来降低蒸腾速率,并且提高瞬时水分利用率,使植物具有更强的抗旱性。QRT-PCR显示PuG7L1的靶基因PuSDD1在PuGTL1-SRDX抑制表达植株和Pu-miR172d过表达植株中均显著上调。综合以上实验结果,说明Pu-miR172d可以抑制PuGTL1进而诱导PuSDD1上调表达来减少气孔密度、降低蒸腾速率、提高水分利用率,从而增强植物的耐旱性,阐明了大青杨Pu-miR172d通过抑制其靶基因PuGTL1的表达来调控植物气孔发育的分子机理。这将为今后研究林木miRNA调节气孔发育及抗旱育种奠定一定的理论基础。
Abstract
qi kong shi zhi wu biao pi you liang ge bao wei xi bao wei rao xing cheng de xiao kong ,zai diao jie zhi wu guang ge gu tan he shui fen san shi de ping heng fang mian ying xiang po da 。qi kong de fa sheng shi yi chong fu za de sheng wu xue guo cheng ,shou duo chong ji yin suo diao kong 。qi kong de fa yo bu dan ke yi ying xiang zhi wu de sheng chang ,ye guan ji dao zhi wu ying dui huan jing xie pai de neng li 。mu qian ,yi you yi xie bao dao fa xian miRNAke yi can yu dao qi kong de fa sheng guo cheng ,dan ji fen zi zuo yong ji zhi reng ran bu shi hen qing xi 。ben yan jiu zhi zai chu bu chan ming da qing yang miR172diao kong qi kong fa yo xiang ying gan han xie pai de fen zi ji li 。li yong sheng wu hua xue 、fen zi sheng wu xue he ji yin gong cheng deng shou duan dui da qing yang Pu-miR172dji ji ba ji yin PuGTL1de fen zi gong neng jin hang le fen xi jian ding 。(1)xiao RNAzhuai lu zu shu ju zhong ba ji yin yu ce jie guo fa xian Trihelixji yin jia zu cheng yuan PuGTL1shi Pu-miR172dde ba ji yin 。li yong 5’RLM-RACEji shu ji nong gan jun shun shi zhuai hua shi yan yan zheng le PuGTL1que shi wei Pu-miR172dde ba ji yin 。shou ci zai da qing yang zhong fa xian le Pu-miR 72ke yi jie ge Trihelixjia zu de zhuai lu yin zi ;(2)li yong sheng wu xin xi xue fang fa he qRT-PCRji shu dui mao guo yang Trihelixzhuai lu yin zi jia zu jin hang chu bu de fen lei ji gong neng jian ding 。Trihelixjia zu you 56ge cheng yuan gou cheng ,ke fen wei wu ge ya zu 。ji zhong ,da duo shu ji yin dou ke yi xiang ying shen tou xie pai 。exImagefen xi jie guo biao ming PuGTL1zhu yao zai you xie zhong biao da 。QRT-PCRjie guo xian shi PuGTL1xia diao biao da zai yi ding cheng du shang ke yi di gao zhi wu kang ni xing ;(3)QRT-PCRjie guo xian shi Pu-miRI72dzai di 1-4pian you xie zhong biao da liang jiao gao ,bing ju ji ke yi xiang ying shen tou xie pai 。ke long da qing yang Pu-miR172dqi dong zi xu lie ,gou jian Pu-miR172dpro::GUSbiao da zai ti 。GUSran se hou fa xian Pu-miR172dzhu yao zai xie pian qi kong zhong biao da ,an shi zhao Pu-miR172dke neng can yu zao ji de qi kong yun dong huo fa yo ;(4)ke long quan chang da qing yang Pu-miR172dxu lie ,gou jian Pu-miR172dguo biao da he yi zhi biao da zai ti 。zu pei miao PEG6000shen tou xie pai hou fa xian ,Pu-miR172dguo biao da zhuai ji yin da qing yang yu WTxiang bi ,chang shi liang hao ju sheng gen lv jiao gao ,er Pu-miR172dyi zhi biao da zhuai ji yin da qing yang yu WTcha bie bu da 。DAB、NBThe Evans bulezu zhi hua xue ran se ji SOD、POD、MDA、dian dao lv he xie lu su han liang deng sheng li zhi biao ce ding jie guo xian shi Pu-miR172dguo biao da zhi zhu de kang ni xing jiao jiang ,shui ming Pu-miR172dju you yi ding de kang shen tou xie pai neng li ;(5)Pu-miR172dguo biao da da qing yang zhi zhu bian ai ,qi kong mi du xian zhe jian shao ,ju jing guang ge su lv 、qi kong dao du he zheng teng su lv jun di yu WT,er shun shi shui fen li yong lv gao yu WT。Pu-miR172dguo biao da zhi zhu zai gan han xie pai xia xie pian han shui liang geng gao ,ju gan han fu shui hou ,Pu-miR172dguo biao da zhi zhu ke ji xu zheng chang sheng chang ,er WThe Pu-miR172dyi zhi biao da zhi zhu sheng chang shou dao yan chong yi zhi 。shou ci fa xian le Pu-miR172dke yi ying xiang qi kong fa yo ,ji ke yi tong guo jian shao qi kong mi du ,jiang di zheng teng su lv ,di gao shun shi shui fen li yong lv ,cong er di gao zhi wu de kang han neng li ;(6)WThe Pu-miR172dguo biao da da qing yang gan han chu li qian hou de zhuai lu zu shu ju zhong ,you da liang de yu guang ge zuo yong 、shi shui xie pai he shen tou xie pai xiang ying de cha yi ji yin 。gan han xie pai hou ,Pu-miR172dyou cu jin le yi xie kang han ji yin biao da 。QRT-PCRjie guo xian shi ,PuGTL1zai Pu-miR172dguo biao da zhi zhu zhong de zhuai lu shui ping ming xian xia jiang ,jin yi bu shui ming le PuGTL shi Pu-miR72dde ba ji yin 。dan PuGTL1ju you zu zhi biao da te yi xing ,zhi zai zi shang er xia de di 1-5pian xie cha yi biao da ;(7)da qing yang PuGTL1-SRDXyi zhi biao da zhuai ji yin da qing yang tu pei miao yu Pu-miR172dguo biao da zhi zhu zai biao xing guan cha he sheng li zhi biao ce ding jie guo fang mian ji ben yi zhi ,PuGTLde yi zhi biao da ye ke shi qi kong mi du jian shao lai jiang di zheng teng su lv ,bing ju di gao shun shi shui fen li yong lv ,shi zhi wu ju you geng jiang de kang han xing 。QRT-PCRxian shi PuG7L1de ba ji yin PuSDD1zai PuGTL1-SRDXyi zhi biao da zhi zhu he Pu-miR172dguo biao da zhi zhu zhong jun xian zhe shang diao 。zeng ge yi shang shi yan jie guo ,shui ming Pu-miR172dke yi yi zhi PuGTL1jin er you dao PuSDD1shang diao biao da lai jian shao qi kong mi du 、jiang di zheng teng su lv 、di gao shui fen li yong lv ,cong er zeng jiang zhi wu de nai han xing ,chan ming le da qing yang Pu-miR172dtong guo yi zhi ji ba ji yin PuGTL1de biao da lai diao kong zhi wu qi kong fa yo de fen zi ji li 。zhe jiang wei jin hou yan jiu lin mu miRNAdiao jie qi kong fa yo ji kang han yo chong dian ding yi ding de li lun ji chu 。
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论文作者分别是来自东北林业大学的王展超，发表于刊物东北林业大学2019-11-18论文，是一篇关于大青杨论文,干旱论文,气孔论文,东北林业大学2019-11-18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，资料来自东北林业大学2019-11-18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：大青杨论文; 干旱论文; 气孔论文; 东北林业大学2019-11-18论文;