本文關(guān)鍵詞： 玉米 抗倒伏 CPS1 AtLOS5 抗鹽 ABA　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：我國是世界上玉米第二大生產(chǎn)國,玉米在我國國民經(jīng)濟發(fā)展中起到非常重要的作用。目前由于氣候條件變化以及灌溉不合理等原因,使得玉米倒伏現(xiàn)象越來越嚴(yán)重,同時大面積的土壤鹽漬化導(dǎo)致耕地質(zhì)量下降,因此提高玉米抗倒伏與抗鹽性是目前急需解決的問題。一方面,在玉米栽培中,提高玉米抗倒伏目前應(yīng)用最多的生長延緩劑是乙烯利,但是乙烯利的使用會造成玉米嚴(yán)重減產(chǎn)。赤霉素在調(diào)節(jié)節(jié)間生長過程中起到很關(guān)鍵的作用,且CPS是合成赤霉素的第一個關(guān)鍵酶。因此本論文對玉米CPS1進行研究,為通過調(diào)控CPS1來塑造玉米理想株型奠定堅實的基礎(chǔ);另一方面,ABA是重要的逆境信號轉(zhuǎn)導(dǎo)植物激素,對玉米響應(yīng)鹽漬化等非生物脅迫具有重要的調(diào)節(jié)作用。在鹽脅迫下,超表達AtLOS5基因能調(diào)控轉(zhuǎn)基因植株一系列的生理生化反應(yīng),從而使其表現(xiàn)出抗鹽性。主要結(jié)果如下:(1)本研究成功從玉米花粉中克隆ZmCPS1基因,全長為2568bp,編碼855個氨基酸,包含DXDD底物催化位點,與其它物種CPSs的氨基酸序列進行比對,同源性大約為50%;該基因在擬南芥和煙草中都定位于質(zhì)體;本研究構(gòu)建了超表達35S::ZmCPS1表達載體,同時轉(zhuǎn)化到野生型擬南芥和cps突變體擬南芥ga1-1和ga1-5中,RT-PCR分析表明ZmCPS1均能可在野生型擬南芥和突變體擬南芥中正常表達,Ler::ZmCPS1株系與野生型Ler相比表型沒有顯著差異,ga1-1::ZmCPS1和ga1-5::ZmCPS1株系均能完全恢復(fù)突變體到野生型表型;本研究利用體外細胞工程的方法成功表達并鑒定出ZmCPS1蛋白信號肽為前面的112aa,表達純化出切掉信號肽且具有活性的ZmCPS1蛋白,進行timecoures 和 kinetics 的研究,最終得出 Kcat=0.25405S-1、KM=0.60173μM 與 Ki=162.74μM;本研究通過CRISPR基因編輯技術(shù)成功獲得了玉米cps1突變體,得知CPS1在調(diào)節(jié)玉米節(jié)間伸長起到關(guān)鍵作用,cps1突變體表現(xiàn)出嚴(yán)重矮化的表型,能被外源的GA恢復(fù)表型。(2)本研究明確了超表達AtLOS5轉(zhuǎn)基因玉米株系的抗鹽性:在鹽脅迫下各轉(zhuǎn)基因株系中AtLOS5均能上調(diào)ABA合成基因的表達與關(guān)鍵酶AO的活性,從而促進ABA的積累;在鹽脅迫下超表達AtLOS5能通過調(diào)控根中K+、Na+、H+凈流速進而使植株體內(nèi)維持較高的K+/Na+,并且調(diào)控其相關(guān)基因的表達從而表現(xiàn)出抗鹽性;在鹽脅迫下超表達AtLOS5能夠維持細胞內(nèi)較高的水勢和滲透勢并緩和根導(dǎo)水率的下降,調(diào)控ZmPIP家族基因的表達使植株體內(nèi)保持較好的水分狀況從而表現(xiàn)出抗鹽性。
[Abstract]:China is the second largest producer of maize in the world, maize plays a very important role in the development of our national economy. The phenomenon of maize lodging is becoming more and more serious, and a large area of soil salinization leads to the decline of cultivated land quality. Therefore, it is urgent to solve the problem of improving the lodging and salt resistance of maize. On the one hand, in maize cultivation. Ethephon is the most widely used growth retarder to improve the lodging resistance of maize. However, the use of ethephon will lead to a serious reduction in maize production. Gibberellin plays a key role in regulating the growth of internode. CPS is the first key enzyme to synthesize gibberellin. Therefore, the study of maize CPS1 in this paper will lay a solid foundation for shaping the ideal plant type of maize by regulating CPS1. On the other hand, ABA is an important stress signal transduction plant hormone, which plays an important role in regulating maize response to abiotic stress, such as salinization. Overexpression of AtLOS5 gene can regulate a series of physiological and biochemical responses of transgenic plants. The main results are as follows: 1) the ZmCPS1 gene was cloned successfully from maize pollen. The total length of ZmCPS1 gene was 2568 BP, encoding 855 amino acids. The amino acid sequence of CPSs of other species was compared with the catalytic site of DXDD substrate, and the homology was about 50; The gene is located in plastids in Arabidopsis thaliana and tobacco. In this study, the overexpression vector of 35s:: ZmCPS1 was constructed and transformed into wild-type Arabidopsis thaliana and cps mutants Arabidopsis thaliana ga1-1 and ga1-5. RT-PCR analysis showed that ZmCPS1 could be expressed normally in wild type Arabidopsis thaliana and mutant Arabidopsis thaliana. There was no significant difference in phenotype between wild type Ler and wild type Ler. Both ga1-1::ZmCPS1 and ga1-5::ZmCPS1 lines could completely restore the mutant to wild-type phenotype. In this study, we successfully expressed and identified the signal peptide of ZmCPS1 protein as 112aa in vitro by cell engineering in vitro, and expressed and purified the ZmCPS1 protein with the activity of cut off signal peptide. Timecoures and kinetics were studied and finally Kcat=0.25405S-1 was obtained. KM=0.60173 渭 M and Ki=162.74 渭 M; In this study, cps1 mutants of maize were successfully obtained by CRISPR gene editing technique, and it was found that CPS1 plays a key role in regulating internode elongation of maize. Cps1 mutants showed severe dwarfing phenotype. Phenotypic recovery by exogenous GA. In this study, we confirmed the salt tolerance of transgenic maize lines with overexpression of AtLOS5: under salt stress, AtLOS5 could up-regulate the expression of ABA synthesis gene and the activity of key enzyme AO. In order to promote the accumulation of ABA; The overexpression of AtLOS5 under salt stress could maintain a high K / Na in the plant by regulating the net flow rate of K ~ (2 +) Na in the root. And the expression of its related genes was regulated to show salt resistance. Under salt stress, overexpression of AtLOS5 could maintain high water potential and osmotic potential in the cells and alleviate the decrease of root water conductivity. Regulating the expression of ZmPIP family genes made the plants maintain good water status and showed salt resistance.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S513

【參考文獻】

相關(guān)期刊論文 前10條

1 李建國;濮勵杰;朱明;張潤森;;土壤鹽漬化研究現(xiàn)狀及未來研究熱點[J];地理學(xué)報;2012年09期

2 李景芳;;淺談玉米倒伏成因及預(yù)防對策[J];種業(yè)導(dǎo)刊;2012年09期

3 程富麗;杜雄;劉夢星;靳小利;崔彥宏;;玉米倒伏及其對產(chǎn)量的影響[J];玉米科學(xué);2011年01期

4 李國恒;李紹偉;吳欣;趙國建;李文倉;劉素玲;;玉米倒伏的原因分析及防治措施[J];中國農(nóng)技推廣;2010年03期

5 戚孝彬;張琳娜;;玉米倒伏的原因及防止對策[J];現(xiàn)代農(nóng)業(yè)科技;2009年11期

6 朱云林;王偉中;顧大路;;化控劑烯世寶對玉米制種產(chǎn)量的影響[J];種子;2007年07期

7 汪寶卿;李召虎;段留生;翟志席;;冠菌素對玉米幼苗耐干旱脅迫的誘導(dǎo)效應(yīng)[J];西北植物學(xué)報;2007年04期

8 葉梅榮;朱昌華;甘立軍;夏凱;;激素間相互作用對植物莖伸長生長的調(diào)控綜述[J];中國農(nóng)學(xué)通報;2007年04期

9 王素平;郭世榮;胡曉輝;賈永霞;焦彥生;;NaCl脅迫對黃瓜幼苗體內(nèi)K~+、Na~+和Cl~-分布的影響[J];生態(tài)學(xué)雜志;2007年03期

10 湯華;柳曉磊;;鹽脅迫下玉米苗期農(nóng)藝性狀和脯氨酸含量變化的研究[J];中國農(nóng)學(xué)通報;2007年03期

，

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