本文主要研究内容
作者田茂竹（2019）在《应用CRISPR/Cas9技术定向突变烟草CYP71D16基因的研究》一文中研究指出：烟草(Nicotiana tabacum L.)是一种重要的经济作物,在我国种植范围较广。烟草品种是影响烟叶产量和品质的主要因素。目前我国对烟草品种的选育仍停留在以表型选择为主的传统育种模式,这种经验育种方式不仅周期长而且品种改良进度缓慢,难以满足我国的经济发展需求。CRISPR/Cas9技术是继锌指核酸酶(ZFNs)、转录激活因子效应物核酸酶(TALENs)技术之后的一种新的基因组编辑技术,它具有方便、高效、周期短以及载体结构简单等优点,这为改良品种以及提高烟叶品质提供了一条简单有效的基因突变技术手段。为快速获得定向突变材料和提高烟草育种效率,因此,本研究应用CRISPR/Cas9技术构建对烟草CYP71D16基因进行定向突变的重组载体,利用该载体遗传转化烟草K326,采用多种方法对转化株进行筛选及突变鉴定,构建并优化了烟草CRISPR/Cas9基因编辑技术体系,获得了定向突变烟草CYP71D16基因的突变株。主要研究结果如下:1.CRISPR/Cas9重组载体构建:通过对烟草CYP71D16基因序列分析,在外显子上选择符合要求的靶点(分别记作SG39、SG38),将靶点与CRISPR/Cas9载体系统连接,构建得到两个重组载体。重组载体采用U6启动子启动sgRNA,采用加强型CaMV35启动子高效表达Cas9蛋白,采用CaMV35S启动子表达潮霉素抗性基因,通过目标序列测序和农杆菌菌液PCR鉴定来验证目标序列的连接和载体的完整性,结果表明构建的载体有很好的完整性,两个重组载体均已导入农杆菌细胞中,可用于后续研究。2.重组载体的遗传转化体系优化:采用根癌农杆菌介导的叶盘转化法开展遗传转化实验,对遗传转化关键环节的技术措施进行了优化。结果表明:(1)潮霉素的最佳抗性筛选浓度是20 mg/L,处于该浓度下的野生型外植体几乎不分化,而且在一段时间后全部褐化死亡。(2)在农杆菌活化培养阶段,采用H1划线方法(平行+均匀交叉)分离的单菌落数量最多,而且大部分都独立地分布在一条直线上,可排除杂菌的污染从而保证单菌落的可靠性。(3)在潮霉素筛选培养阶段,添加100 mg/L替门汀能够大幅度降低侵染后烟草叶片的污染率,高于该浓度虽可完全抑制农杆菌和其他杂菌生长,但同时也对叶片造成了伤害。(4)在脱分化及再生阶段,6-BA和NAA配比为1.0/0.1时更有利于愈伤组织诱导,配比为0.5/0.1时更有利于不定芽分化,两种配比较能同时兼顾愈伤组织诱导和不定芽分化诱导。(5)在生根阶段,采用IAA和IBA配比为0.2/0.2处理的生根量较多,达到34条,平均根长最长,达到3.23 cm,而且根较粗、叶色正常、整体长势好。3.阳性株筛选与突变效应鉴定:采用PCR方法对转化株进行潮霉素抗性基因及Cas9基因筛选鉴定,潮霉素PCR鉴定得到52株阳性株,Cas9基因鉴定得到的阳性株为46株,阳性率达到82.14%,表明有11.54%的Cas9基因在整合过程中丢失或沉默,为了不影响筛选结果的准确性,建议采用Cas9基因PCR检测对阳性株进行筛选。另外,采用一代测序和RT-qPCR方法对阳性株目的基因突变效应进行鉴定,测序结果得出39株阳性株的靶点及附近发生了不同程度的单碱基与氨基酸变异,总编辑效率达到84.78%;RT-qPCR方法鉴定结果得出共38株阳性突变株的CYP71D16基因相对表达量比野生型低,目的基因突变的植株率达到82.61%。鉴定结果说明两种方法有差异,可能的原因是一代测序方法检测的是目的基因序列是否发生改变,而RT-qPCR方法鉴定的是基因表达量的改变,基因序列发生了改变不一定就会导致目的基因在表达上会有所改变,因此在进行突变体鉴定时可两种方法配合使用,也可以只用后者进行鉴定。本研究应用CRISPR/Cas9技术定向突变烟草CYP71D16基因,获得了38份突变材料,目的基因突变的植株率达到82.61%。说明CRISPR/Cas9基因编辑技术的突变效率高,本研究构建的CRISPR/Cas9基因编辑技术体系达到定向突变烟草基因的实用要求。
Abstract
yan cao (Nicotiana tabacum L.)shi yi chong chong yao de jing ji zuo wu ,zai wo guo chong zhi fan wei jiao an 。yan cao pin chong shi ying xiang yan xie chan liang he pin zhi de zhu yao yin su 。mu qian wo guo dui yan cao pin chong de shua yo reng ting liu zai yi biao xing shua ze wei zhu de chuan tong yo chong mo shi ,zhe chong jing yan yo chong fang shi bu jin zhou ji chang er ju pin chong gai liang jin du huan man ,nan yi man zu wo guo de jing ji fa zhan xu qiu 。CRISPR/Cas9ji shu shi ji xin zhi he suan mei (ZFNs)、zhuai lu ji huo yin zi xiao ying wu he suan mei (TALENs)ji shu zhi hou de yi chong xin de ji yin zu bian ji ji shu ,ta ju you fang bian 、gao xiao 、zhou ji duan yi ji zai ti jie gou jian chan deng you dian ,zhe wei gai liang pin chong yi ji di gao yan xie pin zhi di gong le yi tiao jian chan you xiao de ji yin tu bian ji shu shou duan 。wei kuai su huo de ding xiang tu bian cai liao he di gao yan cao yo chong xiao lv ,yin ci ,ben yan jiu ying yong CRISPR/Cas9ji shu gou jian dui yan cao CYP71D16ji yin jin hang ding xiang tu bian de chong zu zai ti ,li yong gai zai ti wei chuan zhuai hua yan cao K326,cai yong duo chong fang fa dui zhuai hua zhu jin hang shai shua ji tu bian jian ding ,gou jian bing you hua le yan cao CRISPR/Cas9ji yin bian ji ji shu ti ji ,huo de le ding xiang tu bian yan cao CYP71D16ji yin de tu bian zhu 。zhu yao yan jiu jie guo ru xia :1.CRISPR/Cas9chong zu zai ti gou jian :tong guo dui yan cao CYP71D16ji yin xu lie fen xi ,zai wai xian zi shang shua ze fu ge yao qiu de ba dian (fen bie ji zuo SG39、SG38),jiang ba dian yu CRISPR/Cas9zai ti ji tong lian jie ,gou jian de dao liang ge chong zu zai ti 。chong zu zai ti cai yong U6qi dong zi qi dong sgRNA,cai yong jia jiang xing CaMV35qi dong zi gao xiao biao da Cas9dan bai ,cai yong CaMV35Sqi dong zi biao da chao mei su kang xing ji yin ,tong guo mu biao xu lie ce xu he nong gan jun jun ye PCRjian ding lai yan zheng mu biao xu lie de lian jie he zai ti de wan zheng xing ,jie guo biao ming gou jian de zai ti you hen hao de wan zheng xing ,liang ge chong zu zai ti jun yi dao ru nong gan jun xi bao zhong ,ke yong yu hou xu yan jiu 。2.chong zu zai ti de wei chuan zhuai hua ti ji you hua :cai yong gen ai nong gan jun jie dao de xie pan zhuai hua fa kai zhan wei chuan zhuai hua shi yan ,dui wei chuan zhuai hua guan jian huan jie de ji shu cuo shi jin hang le you hua 。jie guo biao ming :(1)chao mei su de zui jia kang xing shai shua nong du shi 20 mg/L,chu yu gai nong du xia de ye sheng xing wai zhi ti ji hu bu fen hua ,er ju zai yi duan shi jian hou quan bu he hua si wang 。(2)zai nong gan jun huo hua pei yang jie duan ,cai yong H1hua xian fang fa (ping hang +jun yun jiao cha )fen li de chan jun la shu liang zui duo ,er ju da bu fen dou du li de fen bu zai yi tiao zhi xian shang ,ke pai chu za jun de wu ran cong er bao zheng chan jun la de ke kao xing 。(3)zai chao mei su shai shua pei yang jie duan ,tian jia 100 mg/Lti men ting neng gou da fu du jiang di qin ran hou yan cao xie pian de wu ran lv ,gao yu gai nong du sui ke wan quan yi zhi nong gan jun he ji ta za jun sheng chang ,dan tong shi ye dui xie pian zao cheng le shang hai 。(4)zai tuo fen hua ji zai sheng jie duan ,6-BAhe NAApei bi wei 1.0/0.1shi geng you li yu yu shang zu zhi you dao ,pei bi wei 0.5/0.1shi geng you li yu bu ding ya fen hua ,liang chong pei bi jiao neng tong shi jian gu yu shang zu zhi you dao he bu ding ya fen hua you dao 。(5)zai sheng gen jie duan ,cai yong IAAhe IBApei bi wei 0.2/0.2chu li de sheng gen liang jiao duo ,da dao 34tiao ,ping jun gen chang zui chang ,da dao 3.23 cm,er ju gen jiao cu 、xie se zheng chang 、zheng ti chang shi hao 。3.yang xing zhu shai shua yu tu bian xiao ying jian ding :cai yong PCRfang fa dui zhuai hua zhu jin hang chao mei su kang xing ji yin ji Cas9ji yin shai shua jian ding ,chao mei su PCRjian ding de dao 52zhu yang xing zhu ,Cas9ji yin jian ding de dao de yang xing zhu wei 46zhu ,yang xing lv da dao 82.14%,biao ming you 11.54%de Cas9ji yin zai zheng ge guo cheng zhong diu shi huo chen mo ,wei le bu ying xiang shai shua jie guo de zhun que xing ,jian yi cai yong Cas9ji yin PCRjian ce dui yang xing zhu jin hang shai shua 。ling wai ,cai yong yi dai ce xu he RT-qPCRfang fa dui yang xing zhu mu de ji yin tu bian xiao ying jin hang jian ding ,ce xu jie guo de chu 39zhu yang xing zhu de ba dian ji fu jin fa sheng le bu tong cheng du de chan jian ji yu an ji suan bian yi ,zong bian ji xiao lv da dao 84.78%;RT-qPCRfang fa jian ding jie guo de chu gong 38zhu yang xing tu bian zhu de CYP71D16ji yin xiang dui biao da liang bi ye sheng xing di ,mu de ji yin tu bian de zhi zhu lv da dao 82.61%。jian ding jie guo shui ming liang chong fang fa you cha yi ,ke neng de yuan yin shi yi dai ce xu fang fa jian ce de shi mu de ji yin xu lie shi fou fa sheng gai bian ,er RT-qPCRfang fa jian ding de shi ji yin biao da liang de gai bian ,ji yin xu lie fa sheng le gai bian bu yi ding jiu hui dao zhi mu de ji yin zai biao da shang hui you suo gai bian ,yin ci zai jin hang tu bian ti jian ding shi ke liang chong fang fa pei ge shi yong ,ye ke yi zhi yong hou zhe jin hang jian ding 。ben yan jiu ying yong CRISPR/Cas9ji shu ding xiang tu bian yan cao CYP71D16ji yin ,huo de le 38fen tu bian cai liao ,mu de ji yin tu bian de zhi zhu lv da dao 82.61%。shui ming CRISPR/Cas9ji yin bian ji ji shu de tu bian xiao lv gao ,ben yan jiu gou jian de CRISPR/Cas9ji yin bian ji ji shu ti ji da dao ding xiang tu bian yan cao ji yin de shi yong yao qiu 。
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论文作者分别是来自贵州大学的田茂竹，发表于刊物贵州大学2019-07-16论文，是一篇关于烟草论文,遗传转化论文,鉴定论文,贵州大学2019-07-16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，资料来自贵州大学2019-07-16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：烟草论文; 遗传转化论文; 鉴定论文; 贵州大学2019-07-16论文;