本文主要研究内容
作者季喜梅（2019）在《光核桃AmRad23d基因功能的初步研究》一文中研究指出：光核桃(Aygdalus wira Koehne)又叫“康布”(藏语),是蔷薇科桃属落叶乔木,原产中国西藏,具有耐干旱、耐贫瘠和抗病害等特征,是我国特有野生桃种,寿命可达千年,为我国Ⅱ级保护植物,集观赏、药用和食用等多用途于一身,具有较高的生态效益和经济价值。本研究克隆了西藏光核桃DNA修复蛋白基因(radiation sensitivity protein-23,Rad23),研究了光核桃干旱胁迫条件下AnzRad23d基因的表达模式;同时对AmRad23d基因进行生物信息学预测及亚细胞定位;构建了原核表达载体对AmRad23d蛋白进行体外表达与纯化;同时构建了植物过表达载体对拟南芥进行了异源表达研究。结果如下:1.利用3月龄光核桃为试验材料,克隆了AmRad23 基因,分析了干旱胁迫下AmRad23d基因在光核桃叶片和根中的表达模式,发现:干旱胁迫下,AmRad23d基因表达量增高,尤其在叶片中表达量较高。2.对克隆成功的光核桃AmRad23d基因进行生物信息学预测,发现:AmRad23d基因的开放阅读框序列(ORF)全长为1158bp,编码385个氨基酸,蛋白质相对分子质量大小预测为42.3kDa,理论等电点(pI)值为4.69。AmRad23d蛋白为亲水性稳定蛋白且无信号肽,与碧桃的亲缘关系最近为99%。UBQ、UBA及STI1功能结构域在物种间较为保守。且AnRad23d基因定位于细胞核。3.成功构建了 pET-15b-AmRad23d原核表达载体,优化了蛋白表达条件并纯化出AmRad23d蛋白,在30℃,IPTG浓度为0.1mmol/L,诱导8h时可诱导出较高浓度的重组蛋白,经纯化脱盐后获得了纯度约95%的重组融合蛋白AmRad23d(25mg)。4.成功构建了 pC1390-AmRad23d过表达载体,并获得了转基因拟南芥T3代转基因株系OE2、OE3和OE8。过表达AmRad23d基因使拟南芥在非生物胁迫试验中具有更高存活率,提高了拟南芥的抗逆性。干旱控水实验表明过表达拟南芥植株叶片的H202和O2-含量低于野生型拟南芥,表明过表达拟南芥受胁迫损伤程度较低。同时在过表达拟南芥中,调控干旱胁迫相关基因及RD29B、KIN2、COR15、COR47和KIN1的表达量在不同时间点显著上调。在外源施加ABA后,过表达拟南芥中AmRa23d基因表达显著增高,过表达拟南芥发芽率和根长显著高于野生型,表明AmRad23d基因提高了拟南芥对外源ABA的耐受性,同时,ABA相关基因ABA2、ABI1和DREB2A的表达量显著降低。
Abstract
guang he tao (Aygdalus wira Koehne)you jiao “kang bu ”(cang yu ),shi qiang wei ke tao shu la xie qiao mu ,yuan chan zhong guo xi cang ,ju you nai gan han 、nai pin ji he kang bing hai deng te zheng ,shi wo guo te you ye sheng tao chong ,shou ming ke da qian nian ,wei wo guo Ⅱji bao hu zhi wu ,ji guan shang 、yao yong he shi yong deng duo yong tu yu yi shen ,ju you jiao gao de sheng tai xiao yi he jing ji jia zhi 。ben yan jiu ke long le xi cang guang he tao DNAxiu fu dan bai ji yin (radiation sensitivity protein-23,Rad23),yan jiu le guang he tao gan han xie pai tiao jian xia AnzRad23dji yin de biao da mo shi ;tong shi dui AmRad23dji yin jin hang sheng wu xin xi xue yu ce ji ya xi bao ding wei ;gou jian le yuan he biao da zai ti dui AmRad23ddan bai jin hang ti wai biao da yu chun hua ;tong shi gou jian le zhi wu guo biao da zai ti dui ni na gai jin hang le yi yuan biao da yan jiu 。jie guo ru xia :1.li yong 3yue ling guang he tao wei shi yan cai liao ,ke long le AmRad23 ji yin ,fen xi le gan han xie pai xia AmRad23dji yin zai guang he tao xie pian he gen zhong de biao da mo shi ,fa xian :gan han xie pai xia ,AmRad23dji yin biao da liang zeng gao ,you ji zai xie pian zhong biao da liang jiao gao 。2.dui ke long cheng gong de guang he tao AmRad23dji yin jin hang sheng wu xin xi xue yu ce ,fa xian :AmRad23dji yin de kai fang yue dou kuang xu lie (ORF)quan chang wei 1158bp,bian ma 385ge an ji suan ,dan bai zhi xiang dui fen zi zhi liang da xiao yu ce wei 42.3kDa,li lun deng dian dian (pI)zhi wei 4.69。AmRad23ddan bai wei qin shui xing wen ding dan bai ju mo xin hao tai ,yu bi tao de qin yuan guan ji zui jin wei 99%。UBQ、UBAji STI1gong neng jie gou yu zai wu chong jian jiao wei bao shou 。ju AnRad23dji yin ding wei yu xi bao he 。3.cheng gong gou jian le pET-15b-AmRad23dyuan he biao da zai ti ,you hua le dan bai biao da tiao jian bing chun hua chu AmRad23ddan bai ,zai 30℃,IPTGnong du wei 0.1mmol/L,you dao 8hshi ke you dao chu jiao gao nong du de chong zu dan bai ,jing chun hua tuo yan hou huo de le chun du yao 95%de chong zu rong ge dan bai AmRad23d(25mg)。4.cheng gong gou jian le pC1390-AmRad23dguo biao da zai ti ,bing huo de le zhuai ji yin ni na gai T3dai zhuai ji yin zhu ji OE2、OE3he OE8。guo biao da AmRad23dji yin shi ni na gai zai fei sheng wu xie pai shi yan zhong ju you geng gao cun huo lv ,di gao le ni na gai de kang ni xing 。gan han kong shui shi yan biao ming guo biao da ni na gai zhi zhu xie pian de H202he O2-han liang di yu ye sheng xing ni na gai ,biao ming guo biao da ni na gai shou xie pai sun shang cheng du jiao di 。tong shi zai guo biao da ni na gai zhong ,diao kong gan han xie pai xiang guan ji yin ji RD29B、KIN2、COR15、COR47he KIN1de biao da liang zai bu tong shi jian dian xian zhe shang diao 。zai wai yuan shi jia ABAhou ,guo biao da ni na gai zhong AmRa23dji yin biao da xian zhe zeng gao ,guo biao da ni na gai fa ya lv he gen chang xian zhe gao yu ye sheng xing ,biao ming AmRad23dji yin di gao le ni na gai dui wai yuan ABAde nai shou xing ,tong shi ,ABAxiang guan ji yin ABA2、ABI1he DREB2Ade biao da liang xian zhe jiang di 。
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论文作者分别是来自东北林业大学的季喜梅，发表于刊物东北林业大学2019-11-18论文，是一篇关于光核桃论文,干旱论文,功能解析论文,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。
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