【摘要】：大豆(Glycine max L., soybean)是豆科植物,在人類的生產、生活中起著重要的作用。研究表明在廣西地區(qū)影響作物高產的主要因素是酸性土壤,而鋁毒是酸性土壤的主要作用因子。在對大豆中ALMT和MATE基因、谷胱丙肽代謝相關基因以及胼胝質代謝相關基因表達分析時需要用到內參基因作校準。而近幾年的研究表明,沒有任何一個內參基因在所有的實驗條件下都穩(wěn)定表達,因此在實驗中需要對內參基因的穩(wěn)定性進行驗證。本實驗以巴西10號(簡稱BX10)和本地2號(簡稱BD2)大豆為材料,采用1/5 Hoagland水培法對大豆進行0 mmol/L(對照)、0.5mmol/LAlCl3處理,取處理6h、2d和12d的大豆根尖做材料,篩選出鋁毒脅迫下大豆22個內參基因(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)中穩(wěn)定表達的基因,用ACt值分析、GeNorm、NormFinder和BestKeeper這四種方法對內參基因進行分析,結果如下：(1)用△Ct值分析法分析內參基因的穩(wěn)定性,在所有的樣品中MTP基因穩(wěn)定性最好,ABC基因是最不穩(wěn)定的基因；(2)用GeNorm軟件分析內參基因的結果為：在所有的樣本里面最穩(wěn)定的是Peptidase_S10和MTP,最不穩(wěn)定的是UNKl,分別對基因在BX10和BD2品種表達進行分析,結果顯示,在BX10里面最穩(wěn)定的是Peptidase_S10和CYP基因,最不穩(wěn)定的是UNK1,在BD2里面最穩(wěn)定的是RBO和UBC2,最不穩(wěn)定的基因是ABC；(3)用NormFinder軟件分析的結果為：對整體樣本進行分析時結果顯示,UBC2基因在整體樣本中表達最穩(wěn)定；UNKl在整體樣本中的表達是最不穩(wěn)定的。對BX10的樣本進行分析結果發(fā)現(xiàn),MTP基因在BX10樣本中的表達很穩(wěn)定,UNK1基因BX10樣本中的表達最不恒定；對BD2樣本進行分析,結果顯示ELF1A基因在BD2的樣品中表達最穩(wěn)定,Letin基因在BD2中是最不穩(wěn)定的；(4) BestKeeper法分析結果為：在整體樣本中最穩(wěn)定的是Pept_S16穩(wěn)定的,最不穩(wěn)定的是ABC,在BX10里面最穩(wěn)定的是TIF,最不穩(wěn)定的是UNK1,在BD2樣品里面最穩(wěn)定的是F_box,最不穩(wěn)定的是ABC。選用MTP和UBC2這兩個基因作為內參基因校準ALMT和MATE基因、谷胱丙肽代謝相關基因(hGSHS、hPCS、y-ECS、GPX、GR、GST)以及胼胝質代謝相關基因(CalS2、CalS3、CalS5、CalS7、CalS8、CalS9、CalS10、CalS11、CalS12、BG1、 BG2)的表達,結果表明ALMT、GST、CalS7、CalS8等基因在鋁毒脅迫下的表達與對照相比變化明顯,說明這些基因參與了植物的耐鋁機制。以不同的基因為內參時發(fā)現(xiàn),同一個基因的在相同的樣本中的表達結果存在著差異,因此在分析基因表達時使用多個內參基因會使結果更可靠。
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S565.1;X173

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