o2-NILs的構(gòu)建及OPAQUE-2轉(zhuǎn)錄因子與Sh1、Sh2基因的互作研究
發(fā)布時間：2018-11-29 09:38
【摘要】：玉米(Zea mays L.)是我國重要的糧食和飼料作物,在農(nóng)業(yè)生產(chǎn)上發(fā)揮著重要作用。普通玉米由于缺乏賴氨酸、色氨酸等必須氨基酸導致其營養(yǎng)價值偏低。而opaque-2突變基因能大幅度提高籽粒賴氨酸含量,因此選育含o2基因及胚乳修飾基因且綜合性狀優(yōu)良的優(yōu)質(zhì)蛋白玉米(QPM)成為改良現(xiàn)有玉米品質(zhì)性狀的重要途徑�；亟挥N是選育QPM的重要途徑之一,將o2基因回交導入現(xiàn)有優(yōu)良自交系構(gòu)建o2-NILs能拓寬我國QPM種質(zhì)基礎(chǔ),但該方法在o2-NILs構(gòu)建運用中存在一些問題。本文介紹了本實驗室QPM育種項目開展以來所使用的方法,并對該方法面臨的問題進行討論。另外,前人關(guān)于OPAQUE-2轉(zhuǎn)錄因子所調(diào)節(jié)基因的研究大多集中在醇溶蛋白、氨基酸代謝、植物抗逆等方面,很少涉及到淀粉合成相關(guān)基因,本課題前期利用蛋白質(zhì)組學的研究發(fā)現(xiàn)在創(chuàng)制成功的o2-NILs中SH1、SH2蛋白表達水平存在顯著差異,因此O2基因可能在調(diào)控玉米淀粉合成過程中參與了重要作用。本文擬以o2-NILs為研究材料通過酵母單雜交、qRT-PCR和籽粒淀粉測定實驗初步解析O2轉(zhuǎn)錄因子通過影響SH1、SH2表達進而參與玉米籽粒淀粉合成途徑。具體研究內(nèi)容和結(jié)果如下：1.在課題組前人研究的基礎(chǔ)上,采用SSR分子標記輔助選擇成功構(gòu)建44個普通玉米o2近等基因系及3個糯玉米o2近等基因系。同時發(fā)現(xiàn)普通玉米o2-NILs構(gòu)建全過程可利用phi057標記進行輔助選擇；糯玉米o2-NILs構(gòu)建回交世代使用phi057標記選擇o2基因,自交純合世代則使用phi057和phi027篩選o2wx基因。2.使用酵母單雜交分析02轉(zhuǎn)錄因子對Sh1、Sh2基因啟動子的作用,結(jié)果發(fā)現(xiàn)02蛋白能與Sh1基因啟動子Sh1-1、Sh1-3、Sh1-5和Sh1-9片段發(fā)生互作：02蛋白能與Sh2基因啟動子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)大亞基基因Sh2的轉(zhuǎn)錄情況。結(jié)果表明,在中97-7 o2導入系中,Sh2在14DAP、18DAP和26 DAP的表達量分別為中97-7的1.43、1.89、1.44倍且差異達到顯著水平(P14DAP=0.05、P18DAP=0.05、P26DAP=0.03); CAL58 o2導入系中Sh2在14DAP、20DAP和26 DAP的表達量分別為CAL58的1.84、1.83、1.79倍且差異達到顯著水平(P14DAP=0.02、P22DAP=0.05、P26DAP=0.05)。14 DAP以后糯玉米及普通玉米o2導入系中Sh2基因的轉(zhuǎn)錄水平高于野生型,02轉(zhuǎn)錄因子對Sh2基因起負向調(diào)控作用。4.對糯玉米自交系中97-7、300父本、中501及對應的o2-NILs進行粗淀粉含量分析,結(jié)果表明,中97-7 o2導入系(中97-7/o2)粗淀粉含量(72.16%)高于中97-7(60.99%)且差異達到極顯著水平(P=6.6E-5)；300父本o2導入系(300父本/o2)粗淀粉含量(71.31%)高于300父本(65.72%)且差異達到極顯著水平(P=-8E-5)；中501/o2粗淀粉含量(71.34%)高于中501(67.21%)且差異達到極顯著水平(P=8.4E-4)。5.通過2、3、4結(jié)果：推測02轉(zhuǎn)錄因子是Sh2轉(zhuǎn)錄的阻遏物,野生型中02基因能正常轉(zhuǎn)錄編碼02蛋白并且能結(jié)合到Sh2基因啟動子上,對其進行轉(zhuǎn)錄抑制調(diào)節(jié),最終導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.
【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2
本文編號：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.
【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2
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