ABA受体与紫花苜蓿逆境相关基因表达分析研究

发布时间：2018-05-11 11:41

本文选题：ABA受体 + 紫花苜蓿　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：本文以紫花苜蓿不同品种(三得利、赛迪5、新牧2、WL903、WL712、WL656、WL343、WL168)为试验材料,研究了逆境胁迫对紫花苜蓿ABA受体与逆境相关基因的表达的影响,对ABA受体基因进行了生物信息学分析,并对胁迫后ABA受体表达进行了分析,同时分析了喷施外源ABA后苜蓿生理和ABA受体及逆境基因的表达。这对逆境条件下紫花苜蓿抗性品种的选择提供了指导,也为今后苜蓿ABA受体在逆境中的应用及抗逆育种提供科学依据。1.利用生物信息学方法,对PYL不同基因的理化性质、蛋白结构等进行推测分析。结果显示:PYL 9个基因所编码的相应蛋白分子量都在20-25 kd范围,其酸碱性除Medtr5g083270外都为弱酸,都为疏水性蛋白,其中Medtr4g014460和Medtr5g03050属于疏水性稳定蛋白质,其它都是不稳定疏水性蛋白质,都不含信号肽;除Medtr5g03050和Medtr1g016480无糖基化位点外,其它基因糖基化位点数有所不同,都没有跨膜结构域。PYL多肽链二级结构主要有α-螺旋、无规则卷曲和延伸链构成,PYL三级结构中Medtr3g071740、Medtr5g083270和Medtr4g014460及Medtr8g027805和Medtr3g090980蛋白保守结构域的三级结构模型基本一致,其它蛋白三级结构各有不同。分子系统进化关系显示,Medtr1g028380、Medtr3g090980同源关系较近,这二者又与Medtr8g027805同源关系较近。Medtr3g071740、Medtr5g083270同源关系较近,这二者又与Medtr4g014460、Medtr7g070050同源关系较近。Medtr1g016480、Medtr5g030500同源关系较近。对PYL保守结构域分析,其编码的蛋白质均含有putative hydrophobic ligand binding site、protein interface和gate结构,以及PYL特异性位点的多重结构域。其基因理化性质,蛋白结构的相似性表明它们在调节机体某些方面共同发挥协调作用,通过某种特定的方式参与机体功能的实现。2.利用15%PEG-6000模拟渗透胁迫,4%NaCl模拟盐胁迫,4℃模拟低温胁迫,采用RT-PCR方法对蒺藜苜蓿和不同品种紫花苜蓿ABA受体的表达进行分析。结果如下:15%PEG胁迫下,蒺藜苜蓿中Medtr1g016480和Medtr5g083270的表达量与CK相比显著升高,分别提高了678.3%、99.6%、231.3%和611.4%、47.6%、176.7%(P0.05),其它基因变化不显著,紫花苜蓿中WL168的表达量最高,其Medtr1g016480和Medtr5g083270在根茎叶中的表达都为根叶茎(6.10、3.04、1.79和6.51、1.89、1.67)。4%NaCl胁迫下,蒺藜苜蓿Medtr1g016480和Medtr5g083270的表达量与CK相比显著升高,分别提高了781.3%、45.9%、269.8%和558.5%、11.2%、53.6%(P0.05),紫花苜蓿中WL168的表达量最高,其Medtr1g016480和Medtr5g083270在根茎叶中的表达都为根叶茎(6.13、3.01、1.98和5.91、2.63、1.51)。4℃低温胁迫下,蒺藜苜蓿中Medtr1g016480和Medtr5g083270的表达量与CK相比显著升高,分别提高了103.5%、14.3%、474.6%和113.8%、56.6%、557.7%(P0.05),苜蓿品种中WL168基因相对表达量最高,其Medtr1g016480和Medtr5g083270在根茎叶中的表达都为叶根茎(6.12、3.09、1.99和5.23、2.78、1.76)。表明ABA受体表达的主要部位在根和叶中,苜蓿ABA受体Medtr1g016480和Medtr5g083270在ABA调控系统中起重要作用,促进苜蓿抵抗逆境的能力。3.采用50μM的外源ABA喷施正常生长的苜蓿幼苗,连续喷施5天。待处理组叶面发黄,测定处理组与对照组的叶绿素含量和脯氨酸含量,采用RT-PCR方法测其ABA受体表达和逆境基因ORE1、RD29A、SAG12和SAG13的表达水平。结果表明:外源ABA处理下,WL168的叶绿素含量最高,三得利的叶绿素含量最低,TM叶绿素含量显著低于CK,WL168的叶绿素含量下降了37.18%(P0.05);WL168的脯氨酸含量最高,三得利脯氨酸含量最低,TM脯氨酸含量显著高于CK,WL168脯氨酸含量上升了84.0%(P0.05)。外源ABA处理下,苜蓿ABA受体的表达量都显著升高,其中Medtr1g016480、Medtr3g071740、Medtr5g030500、Medtr5g083270和Medtr7g070050的表达量显著高于其它基因。苜蓿品种中WL168基因相对表达量最高,三得利相对表达量最小。逆境相关基因ORE1、RD29A、SAG12和SAG13的相对表达量与CK相比显著提高,各基因表达量由高到低为RD29AORE1SAG13SAG12。其中,表达量最大是WL168,分别提高了431.2%、503.8%、343.6%和405.9%(P0.05);相对表达量最小是三得利,分别提高了282.9%、359.2%、230.8%和261.9%(P0.05);苜蓿可能通过RD29A基因的表达来驱动其它逆境基因使其激活,进而调节植物体完成对逆境胁迫的抵抗。
[Abstract]:In this paper, the effects of adversity stress on the expression of ABA receptor and adversity related genes in alfalfa were studied with different alfalfa varieties (San De Li, sadi 5, new pastoral 2, WL903, WL712, WL656, WL343, WL168). The bioinformatics analysis of the ABA receptor gene was carried out, and the expression of ABA receptor after stress was analyzed, and the analysis of the expression of ABA receptor was also analyzed. The expression of Alfalfa physiology, ABA receptor and adversity gene after spraying exogenous ABA, which provides guidance for the selection of resistant varieties of Alfalfa under adverse conditions, and provides scientific basis for the application of Alfalfa ABA receptor in adversity and resistance breeding,.1. using bioinformatics method, the physicochemical properties and protein structure of different PYL genes. The results show that the molecular weight of the corresponding protein encoded by the 9 genes of PYL is in the range of 20-25 KD, and its acid base is weak acid except Medtr5g083270, and all of them are hydrophobic proteins. Among them, Medtr4g014460 and Medtr5g03050 are hydrophobic and stable proteins, and the others are unstable hydrophobic proteins, neither contain signal peptides; except Med. The number of glycosylation sites of other genes is different from the tr5g03050 and Medtr1g016480 glycosylation sites. There is no two stage structure of.PYL polypeptide chain in the transmembrane domain, including alpha helix, irregular curling and extension chain, and Medtr3g071740, Medtr5g083270 and Medtr4g014460, Medtr8g027805 and Medtr3g090980 protein conserved in PYL three structure. The three level structure model of the structure is basically the same, and the other protein three structure is different. The molecular phylogenetic relationship shows that Medtr1g028380 and Medtr3g090980 have close homology, and these two are close to.Medtr3g071740, Medtr5g083270 homology is closer, and these two are homologous with Medtr4g014460 and Medtr7g070050. The relationship is closer to.Medtr1g016480, and the homology of Medtr5g030500 is close. For the analysis of PYL conservative domain, the encoded proteins contain putative hydrophobic ligand binding site, protein interface and gate structures, and the multiple domains of PYL specific sites. Their genetic properties and the similarity of protein structure indicate that they are in regulation. Some aspects of the body play a coordinated role and participate in the function of the body in a certain way..2. uses 15%PEG-6000 to simulate osmotic stress, 4%NaCl simulates salt stress, 4 degrees centigrade simulated low temperature stress, and RT-PCR method is used to analyze the expression of ABA in alfalfa and alfalfa varieties of different varieties of Tribulus terrestris and different varieties. The results are as follows: 15%PEG stress The expression of Medtr1g016480 and Medtr5g083270 in alfalfa was significantly higher than that of CK, and increased by 678.3%, 99.6%, 231.3% and 611.4%, 47.6%, 176.7% (P0.05), and the other gene changes were not significant. The expression of WL168 in alfalfa was the highest, and the expression of Medtr1g016480 and Medtr5g083270 in the rhizome leaves was the root leaf stem (6.10,3.04,1.). 79 and 6.51,1.89,1.67) under.4%NaCl stress, the expression of Medtr1g016480 and Medtr5g083270 in alfalfa increased significantly compared with CK, and increased by 781.3%, 45.9%, 269.8% and 558.5%, 11.2%, 53.6% (P0.05). The expression of WL168 in alfalfa was the highest, and the expression of Medtr1g016480 and Medtr5g083270 in the rhizome leaves was the root and leaf stem (6.13,3.01,1). .98 and 5.91,2.63,1.51) at.4 C under low temperature stress, the expression of Medtr1g016480 and Medtr5g083270 in alfalfa was significantly higher than that of CK, and increased by 103.5%, 14.3%, 474.6% and 113.8%, 56.6%, 557.7% (P0.05). The relative expression of WL168 gene in alfalfa varieties was the highest, and the expression of Medtr1g016480 and Medtr5g083270 in the rhizome leaves were all leaves. The rhizomes (6.12,3.09,1.99 and 5.23,2.78,1.76) showed that the main sites of ABA receptor expression were in the roots and leaves. Alfalfa ABA receptor Medtr1g016480 and Medtr5g083270 played an important role in the ABA regulation system. The ability to promote alfalfa resistance to adversity,.3. used 50 u M exogenous ABA to spray the normal growth alfalfa seedlings for 5 days. The chlorophyll content and proline content of the treatment group and the control group were measured, and the expression of ABA receptor and adversity gene ORE1, RD29A, SAG12 and SAG13 were measured by RT-PCR method. The results showed that the chlorophyll content of WL168 was the highest, the content of the chlorophyll was the lowest, and the content of TM chlorophyll was significantly lower than CK and WL1. The content of chlorophyll in 68 decreased by 37.18% (P0.05); the content of proline in WL168 was the highest, the content of proline was the lowest, the proline content of TM was significantly higher than that of CK, and the content of WL168 proline increased by 84% (P0.05). The expression of Alfalfa ABA receptor was significantly increased under exogenous ABA treatment, including Medtr1g016480, Medtr3g071740, Medtr5g030500, Medtr5g0832. The expression of 70 and Medtr7g070050 is significantly higher than that of other genes. The relative expression of WL168 gene in alfalfa varieties is the highest, and the relative expression of the three delta is the smallest. The relative expression of the adversity related genes ORE1, RD29A, SAG12 and SAG13 is significantly higher than that of CK, and the expression of each gene is from high to low to RD29AORE1SAG13SAG12., and the maximum expression is WL1 68, 431.2%, 503.8%, 343.6% and 405.9% (P0.05) were increased respectively, and the minimum relative expression was three gain, which increased by 282.9%, 359.2%, 230.8% and 261.9% (P0.05). Alfalfa could activate other adversity genes through the expression of RD29A gene, and then adjust the plant to resist the stress of adversity.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S541.9

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