o2-NILs的构建及OPAQUE-2转录因子与Sh1、Sh2基因的互作研究
发布时间：2018-11-29 09:38
【摘要】：玉米(Zea mays L.)是我国重要的粮食和饲料作物,在农业生产上发挥着重要作用。普通玉米由于缺乏赖氨酸、色氨酸等必须氨基酸导致其营养价值偏低。而opaque-2突变基因能大幅度提高籽粒赖氨酸含量,因此选育含o2基因及胚乳修饰基因且综合性状优良的优质蛋白玉米(QPM)成为改良现有玉米品质性状的重要途径。回交育种是选育QPM的重要途径之一,将o2基因回交导入现有优良自交系构建o2-NILs能拓宽我国QPM种质基础,但该方法在o2-NILs构建运用中存在一些问题。本文介绍了本实验室QPM育种项目开展以来所使用的方法,并对该方法面临的问题进行讨论。另外,前人关于OPAQUE-2转录因子所调节基因的研究大多集中在醇溶蛋白、氨基酸代谢、植物抗逆等方面,很少涉及到淀粉合成相关基因,本课题前期利用蛋白质组学的研究发现在创制成功的o2-NILs中SH1、SH2蛋白表达水平存在显著差异,因此O2基因可能在调控玉米淀粉合成过程中参与了重要作用。本文拟以o2-NILs为研究材料通过酵母单杂交、qRT-PCR和籽粒淀粉测定实验初步解析O2转录因子通过影响SH1、SH2表达进而参与玉米籽粒淀粉合成途径。具体研究内容和结果如下：1.在课题组前人研究的基础上,采用SSR分子标记辅助选择成功构建44个普通玉米o2近等基因系及3个糯玉米o2近等基因系。同时发现普通玉米o2-NILs构建全过程可利用phi057标记进行辅助选择；糯玉米o2-NILs构建回交世代使用phi057标记选择o2基因,自交纯合世代则使用phi057和phi027筛选o2wx基因。2.使用酵母单杂交分析02转录因子对Sh1、Sh2基因启动子的作用,结果发现02蛋白能与Sh1基因启动子Sh1-1、Sh1-3、Sh1-5和Sh1-9片段发生互作：02蛋白能与Sh2基因启动子Sh2-7片段互作。3.分析不同o2近等基因系籽粒胚乳中的ADPG焦磷酸化酶(AGPase)大亚基基因Sh2的转录情况。结果表明,在中97-7 o2导入系中,Sh2在14DAP、18DAP和26 DAP的表达量分别为中97-7的1.43、1.89、1.44倍且差异达到显著水平(P14DAP=0.05、P18DAP=0.05、P26DAP=0.03); CAL58 o2导入系中Sh2在14DAP、20DAP和26 DAP的表达量分别为CAL58的1.84、1.83、1.79倍且差异达到显著水平(P14DAP=0.02、P22DAP=0.05、P26DAP=0.05)。14 DAP以后糯玉米及普通玉米o2导入系中Sh2基因的转录水平高于野生型,02转录因子对Sh2基因起负向调控作用。4.对糯玉米自交系中97-7、300父本、中501及对应的o2-NILs进行粗淀粉含量分析,结果表明,中97-7 o2导入系(中97-7/o2)粗淀粉含量(72.16%)高于中97-7(60.99%)且差异达到极显著水平(P=6.6E-5)；300父本o2导入系(300父本/o2)粗淀粉含量(71.31%)高于300父本(65.72%)且差异达到极显著水平(P=-8E-5)；中501/o2粗淀粉含量(71.34%)高于中501(67.21%)且差异达到极显著水平(P=8.4E-4)。5.通过2、3、4结果：推测02转录因子是Sh2转录的阻遏物,野生型中02基因能正常转录编码02蛋白并且能结合到Sh2基因启动子上,对其进行转录抑制调节,最终导致野生型植株籽粒淀粉含量增加。
[Abstract]:Maize (Zea mays L.) Is our country important grain and fodder crop, plays an important role in the agricultural production. The nutritional value of common maize is low due to lack of lysine, tryptophan and other essential amino acids. However, opaque-2 mutation gene can greatly increase the content of lysine in grain. Therefore, breeding (QPM) with high quality protein containing O2 gene and endosperm modifier gene and excellent synthesis is an important way to improve the quality traits of existing maize. Backcross breeding is one of the important ways to select QPM. Introducing O2 gene backcross into existing excellent inbred lines to construct o2-NILs can broaden the QPM germplasm base in China, but there are some problems in the application of this method in o2-NILs construction. This paper introduces the methods used since the development of QPM breeding project in our laboratory, and discusses the problems faced by this method. In addition, previous studies on genes regulated by OPAQUE-2 transcription factors mostly focus on gliadin, amino acid metabolism, plant stress resistance and so on. At the beginning of this study, we found that there were significant differences in the expression level of SH1,SH2 protein in the successfully created o2-NILs, so O2 gene may play an important role in the regulation of corn starch synthesis. In this paper, o2-NILs was used as a material to study the effects of O _ 2 transcription factors on the starch synthesis of maize grains through yeast single hybrid, qRT-PCR and grain starch determination. The effect of O _ 2 transcription factors on the expression of O _ 2 was studied. The specific research contents and results are as follows: 1. On the basis of previous studies, 44 normal maize o2 near-isogenic lines and 3 waxy maize o2 near-isogenic lines were successfully constructed by SSR molecular marker-assisted selection. At the same time, it was found that the whole process of o2-NILs construction in common maize could be selected by phi057 markers, the phi057 marker was used to select O2 gene in backcross generation of waxy maize o2-NILs construction, and phi057 and phi027 were used to screen o2wx gene in self-homozygote generation. The effect of transcription factor 02 on the promoter of Sh1,Sh2 gene was analyzed by yeast single hybrid analysis. The results showed that 02 protein could work with Sh1-1,Sh1-3, of Sh1 gene promoter. The interaction between Sh1-5 and Sh1-9 fragment: 02 protein can interact with Sh2-7 fragment of Sh2 gene promoter. 3. 3. The transcription of ADPG pyrophosphorylase (AGPase) large subunit gene Sh2 in grain endosperm of different o2 near-isogenic lines was analyzed. The results showed that the expression of Sh2 in 14DAPP 18DAP and 26 DAP was 1.44 times higher than that in Zhong97-7o2, respectively, and the difference was significant (P14DAPO0.05P18DAPP 0.05P26DAP0.03). The expression of Sh2 in 14DAPP 20DAP and 26 DAP was 1.79 times of that of CAL58, respectively, and the difference was significant (P14DAPP0.02P22DAPP 0.05). P26DAP=0.05). After 14 DAP, the transcriptional level of Sh2 gene was higher in waxy maize and common maize o2 lines than that in wild type, and the transcription factor 02 played a negative role in regulating Sh2 gene. 4. The crude starch contents of 97-7300 male, Zhong 501 and corresponding o2-NILs in waxy maize inbred lines were analyzed. The crude starch content (72.16%) of the middle 97-7 O2 line (middle 97-7/o2) was higher than that of the middle 97-7 line (60.99%), and the difference reached a very significant level (P=6.6E-5). The crude starch content (71.31%) of the imported line (300male / O2) was higher than that of the 300male parent (65.72%), and the difference reached a very significant level (P=-8E-5). The crude starch content of medium 501/o2 (71.34%) was higher than that of Zhong501 (67.21%), and the difference was very significant (P=8.4E-4). The results showed that the 02 transcription factor was a repressor of Sh2 transcription, and that the 02 gene in wild type could encode 02 protein normally and bind to the promoter of Sh2 gene to regulate its transcription inhibition. Finally, the starch content of wild type plants increased.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：2364642
[Abstract]:Maize (Zea mays L.) Is our country important grain and fodder crop, plays an important role in the agricultural production. The nutritional value of common maize is low due to lack of lysine, tryptophan and other essential amino acids. However, opaque-2 mutation gene can greatly increase the content of lysine in grain. Therefore, breeding (QPM) with high quality protein containing O2 gene and endosperm modifier gene and excellent synthesis is an important way to improve the quality traits of existing maize. Backcross breeding is one of the important ways to select QPM. Introducing O2 gene backcross into existing excellent inbred lines to construct o2-NILs can broaden the QPM germplasm base in China, but there are some problems in the application of this method in o2-NILs construction. This paper introduces the methods used since the development of QPM breeding project in our laboratory, and discusses the problems faced by this method. In addition, previous studies on genes regulated by OPAQUE-2 transcription factors mostly focus on gliadin, amino acid metabolism, plant stress resistance and so on. At the beginning of this study, we found that there were significant differences in the expression level of SH1,SH2 protein in the successfully created o2-NILs, so O2 gene may play an important role in the regulation of corn starch synthesis. In this paper, o2-NILs was used as a material to study the effects of O _ 2 transcription factors on the starch synthesis of maize grains through yeast single hybrid, qRT-PCR and grain starch determination. The effect of O _ 2 transcription factors on the expression of O _ 2 was studied. The specific research contents and results are as follows: 1. On the basis of previous studies, 44 normal maize o2 near-isogenic lines and 3 waxy maize o2 near-isogenic lines were successfully constructed by SSR molecular marker-assisted selection. At the same time, it was found that the whole process of o2-NILs construction in common maize could be selected by phi057 markers, the phi057 marker was used to select O2 gene in backcross generation of waxy maize o2-NILs construction, and phi057 and phi027 were used to screen o2wx gene in self-homozygote generation. The effect of transcription factor 02 on the promoter of Sh1,Sh2 gene was analyzed by yeast single hybrid analysis. The results showed that 02 protein could work with Sh1-1,Sh1-3, of Sh1 gene promoter. The interaction between Sh1-5 and Sh1-9 fragment: 02 protein can interact with Sh2-7 fragment of Sh2 gene promoter. 3. 3. The transcription of ADPG pyrophosphorylase (AGPase) large subunit gene Sh2 in grain endosperm of different o2 near-isogenic lines was analyzed. The results showed that the expression of Sh2 in 14DAPP 18DAP and 26 DAP was 1.44 times higher than that in Zhong97-7o2, respectively, and the difference was significant (P14DAPO0.05P18DAPP 0.05P26DAP0.03). The expression of Sh2 in 14DAPP 20DAP and 26 DAP was 1.79 times of that of CAL58, respectively, and the difference was significant (P14DAPP0.02P22DAPP 0.05). P26DAP=0.05). After 14 DAP, the transcriptional level of Sh2 gene was higher in waxy maize and common maize o2 lines than that in wild type, and the transcription factor 02 played a negative role in regulating Sh2 gene. 4. The crude starch contents of 97-7300 male, Zhong 501 and corresponding o2-NILs in waxy maize inbred lines were analyzed. The crude starch content (72.16%) of the middle 97-7 O2 line (middle 97-7/o2) was higher than that of the middle 97-7 line (60.99%), and the difference reached a very significant level (P=6.6E-5). The crude starch content (71.31%) of the imported line (300male / O2) was higher than that of the 300male parent (65.72%), and the difference reached a very significant level (P=-8E-5). The crude starch content of medium 501/o2 (71.34%) was higher than that of Zhong501 (67.21%), and the difference was very significant (P=8.4E-4). The results showed that the 02 transcription factor was a repressor of Sh2 transcription, and that the 02 gene in wild type could encode 02 protein normally and bind to the promoter of Sh2 gene to regulate its transcription inhibition. Finally, the starch content of wild type plants increased.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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