【作者】 夏卓盛；

【导师】 吴跃明； 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2007， 硕士

【摘要】 酸性土壤分布范围广,整治改造困难,且土壤中所含的高浓度铝离子会严重抑制作物根系的生长,导致减产甚至死亡。本研究通过抑制消减杂交(SSH)技术构建了紫花苜蓿铝胁迫正反向文库,并分离克隆与紫花苜蓿铝诱导表达相关基因,初步揭示与紫花苜蓿耐铝相关的基因,为分离和鉴定耐铝关键基因打下了基础。主要研究结果如下：试验一选取前期已筛选出来的紫花苜蓿耐铝品种小冠,采用单因子试验设计,设置不同梯度的铝离子胁迫浓度(0、20、30、40、50、60μM)采用水培法对紫花苜蓿幼苗进行24h胁迫试验,通过测定相对根伸长量(RRE)发现40μM铝离子溶液能完全抑制根的伸长。试验二采用抑制消减杂交(SSH)技术,以铝胁迫的苜蓿为Tester,未胁迫的为Driver,经过总RNA抽提,mRNA纯化,逆转录成cDNA,四碱基酶切和接头连接后,经过两次消减杂交和两次PCR特异性扩增后,经过T-A克隆,蓝白斑筛选等步骤构建一个铝胁迫正向消减文库。以铝胁迫的cDNA为Driver,未胁迫的cDNA为Tester,构建了反向消减文库文库,具体结果如下：1、紫花苜蓿经CaCl2溶液(pH 4.5)培养3d后,试验组用40μM铝离子胁迫处理24h,对照组无铝处理继续培养24h。用Trizol法分别提取幼苗根部总RNA,试验组和对照组的浓度分别达到1801 ng／μl和1812 ng／μl,且纯度较高。2、采用抑制性消减杂交法(SSH)分别构建了一个964个克隆的正向消减文库和一个768个克隆的反向消减文库。菌落PCR显示插入片段在250bp到1200bp之间,文库质量良好。3、从正向消减文库中随机挑选40个克隆进行测序,共获待34个有效序列,其中包括9个新的EST序列(登陆号EH643286-EH643294)和25个与已知基因相关的EST序列,并登录到dbEST数据库。经BLASTX分析表明这些EST序列编码果胶甲脂酶、过氧化物酶、钙依赖蛋白激酶、蔗糖转化酶、蛋白磷酸脂酶调节因子、几丁质酶、核酸结合蛋白、变位酶、甘油磷酸二酯酶、橡胶延伸因子、糖转运蛋白、细胞色素P450、乙酰氨基己糖苷酶等。本试验克隆到一个与耐铝性密切相关的基因,编码果胶甲酯酶。该酶能促进根细胞壁果胶的去甲基化作用,产生游离羧基,从而增加细胞壁上的铝结合位点,减少铝对植物的毒害作用。同时,该酶也是植物根细胞的伸长受到铝毒抑制进而影响根系伸长的可能原因。如何具体评价果胶甲酯酶的上调对紫花苜蓿在抵御铝毒中的作用,以及果胶甲酯酶的表达量与紫花苜蓿耐铝能力的关系,还有待进一步的研究。其他EST所编码的蛋白涉及到抗氧化作用、信号的传导、发育和能量代谢等多种生理过程,与其他生物性和非生物性胁迫诱导相关,广泛参与植物的其它应激反应,属于植物的非特异性应答反应。更多还原

【Abstract】 Acid siol distribute abroad in the world and is difficult to be meliorated. The high concentration of aluminum will greatly inhibit root elongation, reduce the plant production, and probably lead to death. The aim of the current research was using suppression subtractive hybridization(SSH) to construct a forward and reverse cDNA library in order to isolate and clone the aluminum tolerance related genes to look into molecular events associated with aluminum resistance and establish fundamental materials and techniques for further study and genetic selection. The main results abtained are listed bellow:In experiment 1, The most aluminum tolerance alfalfa cultivar xiao guan(Rcent results of laboratory alfalfa tolerance screening experiment) was adopted to evaluate the most suitable aluminum concentration by using single factor test with solution culture method under a gradient aluminum concentration stress (0、20、30、40、50、60uM) for 24h. The result show that 40μM aluminum can completely inhibit the root elongation.In experiment 2, Using the suppression subtractive hybridization(SSH) techology, the alfalfa with aluminum stressed served as tester and without aluminum stre ssed as Driver. By total RNA extraction, mRNA purification, cDNA synthesis, RsaⅠdigestion, adaptor ligation, twice hybridization and twice PCR amplification ect, a forward subtractive library was constructed. Vice versa, when the alfalfa with aluminum stressed served as Driver.and without aluminum stressed as tester, It was a reverse subtractive library. The detail results were below. 1) After germination, seeds were cultivated in CaCl2 solution(pH 4.5) 3d and then divide into two group, one group(Driver) is keep on cultivating in CaCl2 solution and another(Tester) is stressed by 40μM aluminum solution. Collect the root after 24h and extract total RNA by Trizol reagent. The total RNA concentration of Tester and Driver were 1801 ng/μl and 1812 ng/μl separately with a good purity.2) Forward and reverse subtraction were conducted with the method of suppression subtractive hybridization(SSH) by PCR-SelestTM cDNA Subtration Kit(Clontech). A 964 clone forward subtration library and a 768 clone reverse subtration library were constructed. Colony PCR suggested that the iniserts were between 250bp-1200bp and the library was suitable for the following work.3) 40 clones were randomly picked, secquenced and obtained 34 unique ESTs. BLASTX show that 25 ESTs found the homology which were all submitted to genbank. These ESTs code for Pectinesterase, peroxidase, calcium-dependent protein kinase, neutral/alkaline invertas, protein phosphatase regulator, chitinase, nucleic acid binding, Chorismate mutase, glycerophosphoryl diester phosphodiesterase, Rubber elongation factor, cytochrome P450, acetylhexosaminidase ect. One EST coding for Pectinesterase which can catalyse pectin and results in more Al binding sites in root cell wall may play the role in alfalfa aluminum detixitifcation. Meanwhile, this mechanism also is one of the reasons why plant root growth affected by aluminum. The role of Pectinesterase played in alfalfa aluminum detoxification and the relationship of alfalfa aluminum tolerance and expression level need to be future studied. The other ESTs were involed in plant antioxidative stress, signal transduction, development and energy metaboly, and these physiological responses were also abserved to be similar to those occuring under other stresses. Nine ESTs had no homology from NCBI(access number EH643286-EH643294).更多还原