铝毒胁迫下大豆内参基因的筛选及相关基因表达分析

发布时间：2018-12-17 12:04

【摘要】：大豆(Glycine max L., soybean)是豆科植物,在人类的生产、生活中起着重要的作用。研究表明在广西地区影响作物高产的主要因素是酸性土壤,而铝毒是酸性土壤的主要作用因子。在对大豆中ALMT和MATE基因、谷胱丙肽代谢相关基因以及胼胝质代谢相关基因表达分析时需要用到内参基因作校准。而近几年的研究表明,没有任何一个内参基因在所有的实验条件下都稳定表达,因此在实验中需要对内参基因的稳定性进行验证。本实验以巴西10号(简称BX10)和本地2号(简称BD2)大豆为材料,采用1/5 Hoagland水培法对大豆进行0 mmol/L(对照)、0.5mmol/LAlCl3处理,取处理6h、2d和12d的大豆根尖做材料,筛选出铝毒胁迫下大豆22个内参基因(18s rRNA、ABC、 ACLB-2、Actin、CRK、CYP、ELFlA、ELFlB、F-box、G6PDH、Letin、MTP、Pept_S16、 Peptidase_S10、PP2A、RBO、SUBI-2、TIF、TUB、UBC2、UNK1、UNK2)中稳定表达的基因,用ACt值分析、GeNorm、NormFinder和BestKeeper这四种方法对内参基因进行分析,结果如下：(1)用△Ct值分析法分析内参基因的稳定性,在所有的样品中MTP基因稳定性最好,ABC基因是最不稳定的基因；(2)用GeNorm软件分析内参基因的结果为：在所有的样本里面最稳定的是Peptidase_S10和MTP,最不稳定的是UNKl,分别对基因在BX10和BD2品种表达进行分析,结果显示,在BX10里面最稳定的是Peptidase_S10和CYP基因,最不稳定的是UNK1,在BD2里面最稳定的是RBO和UBC2,最不稳定的基因是ABC；(3)用NormFinder软件分析的结果为：对整体样本进行分析时结果显示,UBC2基因在整体样本中表达最稳定；UNKl在整体样本中的表达是最不稳定的。对BX10的样本进行分析结果发现,MTP基因在BX10样本中的表达很稳定,UNK1基因BX10样本中的表达最不恒定；对BD2样本进行分析,结果显示ELF1A基因在BD2的样品中表达最稳定,Letin基因在BD2中是最不稳定的；(4) BestKeeper法分析结果为：在整体样本中最稳定的是Pept_S16稳定的,最不稳定的是ABC,在BX10里面最稳定的是TIF,最不稳定的是UNK1,在BD2样品里面最稳定的是F_box,最不稳定的是ABC。选用MTP和UBC2这两个基因作为内参基因校准ALMT和MATE基因、谷胱丙肽代谢相关基因(hGSHS、hPCS、y-ECS、GPX、GR、GST)以及胼胝质代谢相关基因(CalS2、CalS3、CalS5、CalS7、CalS8、CalS9、CalS10、CalS11、CalS12、BG1、 BG2)的表达,结果表明ALMT、GST、CalS7、CalS8等基因在铝毒胁迫下的表达与对照相比变化明显,说明这些基因参与了植物的耐铝机制。以不同的基因为内参时发现,同一个基因的在相同的样本中的表达结果存在着差异,因此在分析基因表达时使用多个内参基因会使结果更可靠。
[Abstract]:Soybean (Glycine max L. (soybean) is a leguminous plant, which plays an important role in human production and life. The results showed that acid soil was the main factor that affected the high yield of crops in Guangxi, and aluminum toxicity was the main action factor of acid soil. The expression of ALMT and MATE genes, glutathione metabolism-related genes and callose metabolism-related genes in soybean should be calibrated. However, recent studies have shown that none of the internal reference genes are stably expressed under all experimental conditions, so the stability of the internal reference genes should be verified in the experiments. In this experiment, the soybean of Brazil 10 (BX10) and local 2 (BD2) were treated with 0 mmol/L (control) and 0.5mmol/LAlCl3 (1 / 5 Hoagland) for 0 mmol/L (control) and 0.5mmol/LAlCl3, and the root tips of soybean treated for 6 h and 12 d were used as materials. Screening of 22 Internal reference genes (18s rRNA,ABC, ACLB-2,Actin,CRK,CYP,ELFlA,ELFlB,F-box,G6PDH,Letin,MTP,Pept_S16, Peptidase_S10,PP2A,RBO,SUBI-2,TIF,TUB,UBC2,UNK1,) of Soybean under Aluminum toxicity stress The stably expressed genes in UNK2 were analyzed by ACt value analysis, GeNorm,NormFinder and BestKeeper methods. The results were as follows: (1) the stability of internal reference genes was analyzed by Ct value analysis. Among all the samples, MTP gene was the most stable and ABC gene was the most unstable gene. (2) the results of GeNorm software analysis showed that Peptidase_S10 and MTP, were the most stable in all samples. The expression of UNKl, in BX10 and BD2 were analyzed respectively. The most stable genes in BX10 are Peptidase_S10 and CYP, and the most unstable in UNK1, in BD2 is RBO and UBC2,. The most unstable gene is ABC;. (3) the results of NormFinder software analysis show that the expression of UBC2 gene is the most stable in the whole sample, and the expression of UNKl in the whole sample is the most unstable. The results of BX10 analysis showed that the expression of MTP gene in BX10 samples was stable, and the expression of UNK1 gene BX10 samples was the most unstable. The analysis of BD2 samples showed that the expression of ELF1A gene was the most stable in BD2 samples, and Letin gene was the most unstable in BD2. (4) the results of BestKeeper analysis are as follows: in the whole sample, the most stable is Pept_S16, the most unstable is that ABC, is the most stable in BX10, and the most unstable in TIF, is UNK1, in BD2. The most unstable is ABC. Two genes, MTP and UBC2, were used to calibrate ALMT and MATE genes, glutathione metabolism related genes (hGSHS,hPCS,y-ECS,GPX,GR,GST) and corpus callosum metabolism-associated genes (CalS2,CalS3,CalS5,CalS7,CalS8,CalS9,CalS10,CalS11,). The results showed that the expression of ALMT,GST,CalS7,CalS8 and other genes was significantly different from that of the control under aluminum toxicity, indicating that these genes were involved in the mechanism of aluminum tolerance in plants. When different genes are used as internal reference, it is found that there are differences in the expression results of the same gene in the same sample. Therefore, the use of multiple internal reference genes in the analysis of gene expression will make the results more reliable.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S565.1;X173

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