盐胁迫下甜高粱NADP-ME基因的功能分析
发布时间：2018-08-06 14:17
【摘要】：本试验通过分析转录组测序结果推测NADP-ME基因与耐盐性相关,进而从甜高粱叶片中提取NADP-ME基因,转入拟南芥并进行该基因的表达和功能分析。主要试验结果如下:1、为了研究甜高粱的耐盐机理,我们用0 mM和150 mM NaCl处理甜高粱幼苗,提取叶片RNA进行转录组测序。通过KEGG途径分析发现,在M-81E中有150个基因注释到70个通路中,而在罗马中有174个基因注释到63个通路中。M-81E和罗马在盐胁迫下的差异表达基因,主要集中在光合碳固定途径中,并且NADP-ME基因被注释到光合作用途径中。在盐胁迫下,该基因在M-81E中的表达量相当高,然而在罗马中却没有明显变化。我们推测NADP-ME可能通过调节光合效率来影响甜高粱耐盐性,所以从甜高粱中分离了NADP-ME基因,并验证其功能。2、我们根据甜高粱已知NADP-ME序列设计引物。通过PCR的方法从甜高粱叶片中克隆NADP-ME基因,该基因全长1911 bp,编码637个氨基酸,分子质量约为15.7 kDa。经过同源序列比对发现,甜高粱叶片中NADP-ME基因与白茅和玉米的同源性最高,达到98%和96%。3、将NADP-ME基因与pCAMBIA3300载体进行重组,利用花序侵染的方法侵染拟南芥,获得过表达植株。通过Basta筛选标记对过表达株系进行筛选,在DNA水平检测过表达株系。4、在盐胁迫下,野生型拟南芥与过表达株系的萌发率、萌发势和主根长度都受到了抑制,但野生型受到的抑制程度较严重,从试验结果可以看出,在萌发期过表达株系的耐盐能力较好;用RT-PCR检测过表达株系中甜高粱NADP-ME基因在不同盐胁迫下的表达量,结果发现在100 mM NaCl处理下该基因的表达量最高。因此选用0 mM和100 mM NaCl处理拟南芥植株,测定叶片中的光合参数、叶绿素含量、荧光参数和光系统I的活性。结果表明,未经盐处理条件下,过表达株系的光合活性比野生型高,其它生理指标与野生型基本一致。在盐胁迫下,过表达株系的Pn、Ci、Tr以及Gs的含量显著高于野生型拟南芥,说明该基因的表达能够提高拟南芥在盐胁迫下的光合效率。过表达株系的叶绿素含量、ΦPSII、Fv/Fm及ΔI/Io活性显著高于野生型拟南芥,Fo、1-qp和NPQ显著低于野生型拟南芥。这些结果说明在盐胁迫下,过表达株系的光合能力较强。为了进一步研究过表株系的耐盐性,我们又对其生物量进行了测定,在盐胁迫下,过表达株系的鲜重和干重都明显高于野生型。说明过表达株系耐盐程度高于野生型。5、选取SALK_064163(At1g79750),CS855818(At1g79750),SALK_036898(At2g19900),SALK_073818C(At5g11670),CS833585(At5g11670)和SALK_139336C(At5g25880),这六个突变体进行试验,结果发现,SALK_064163和SALK_073818C这两个突变体的耐盐能力显著低于野生型。盐胁迫下,SALK_064163和SALK_073818C这两个突变体株系在萌发期的萌发率、萌发势和主根长度明显低于野生型拟南芥,因此选用了这两个盐敏感突变体进行后续试验。在幼苗期,通过测定各项生理指标发现,在100 mM NaCl处理下,SALK_064163和SALK_073818C的Pn、Ci、Tr和Gs的活性都显著低于野生型拟南芥,叶绿素含量、ΦPSII、Fv/Fm及ΔI/Io活性均显著低于野生型植株,而Fo、1-qp和NPQ显著高于野生型植株,并且这两个突变体的鲜重和干重都明显低于野生型。这些结果表明,在盐胁迫下,SALK_064163和SALK_073818C的光系统受盐害程度高于野生型,并且其耐盐性明显低于野生型。上述结果表明,甜高粱NADP-ME基因能够提高拟南芥幼苗在盐胁迫下的光合能力。
[Abstract]:By analyzing the results of the transcriptional sequence, we speculated that the NADP-ME gene was related to the salt tolerance, and then extracted the NADP-ME gene from the sweet sorghum leaves and transferred to Arabidopsis and carried out the expression and functional analysis of the gene. The main results were as follows: 1, in order to study the salt tolerance mechanism of sweet sorghum, the sweet sorghum seedlings were treated with 0 mM and 150 mM NaCl. The KEGG pathway analysis showed that 150 genes were annotated to 70 pathways in M-81E, and there were 174 gene annotations to 63 pathways in Rome and the differential expression genes under salt stress in 63 pathways, mainly concentrated in the photosynthetic carbon fixation pathway, and the NADP-ME gene was annotated to photosynthesis. Under salt stress, the gene expression in M-81E is quite high, but there is no obvious change in Rome. We speculate that NADP-ME may affect the salt tolerance of sweet sorghum by regulating photosynthetic efficiency, so NADP-ME gene is isolated from sweet sorghum, and its function.2 is verified. We set up a known NADP-ME sequence based on sweet sorghum. Primers. The NADP-ME gene was cloned from sweet sorghum leaves by PCR. The gene was 1911 BP and 637 amino acids were encoded. The molecular weight was about 15.7 kDa.. The homology of the NADP-ME gene in sweet sorghum leaves was found to be the highest, reaching 98% and 96%.3. The NADP-ME gene and pCAMBIA3300 vector were added to the sweet sorghum leaves. The overexpressed plant was infected by the method of inflorescence infection. The overexpressed plant was screened by Basta screening marker and the overexpressed strain.4 was detected at the level of DNA. Under salt stress, the germination rate, germination potential and the length of the main root of the wild Arabidopsis and overexpressed lines were suppressed, but the wild type was inhibited. The test results showed that the salt tolerance of the overexpressed strain was better in the germination period, and the expression amount of the sweet sorghum NADP-ME gene under the different salt stress was detected by RT-PCR. The results showed that the expression of the gene was the highest under the treatment of 100 mM NaCl. Therefore, 0 mM and 100 mM NaCl were selected for the treatment of Arabidopsis plants. Test the photosynthetic parameters, chlorophyll content, fluorescence parameters and the activity of I in the light system. The results showed that the photosynthetic activity of overexpressed lines was higher than that of wild type without salt treatment, and the other physiological indexes were basically the same as those of wild type. Under salt stress, the content of Pn, Ci, Tr and Gs in overexpressed lines was significantly higher than that of wild Arabidopsis. The expression of this gene could improve the photosynthetic efficiency of Arabidopsis under salt stress. The content of chlorophyll, PSII, Fv/Fm and delta I/Io of overexpressed lines were significantly higher than that of wild Arabidopsis, and Fo, 1-qp and NPQ were significantly lower than those of wild Arabidopsis. These results indicated that the photosynthetic capacity of overexpressed lines was stronger under salt stress. We also studied the salt tolerance of the plant lines. Under salt stress, the fresh weight and dry weight of overexpressed lines were significantly higher than those of the wild type. It showed that the salt tolerance of overexpressed lines was higher than that of wild type.5, SALK_064163 (At1g79750), CS855818 (At1g79750), SALK_036898 (At2g19900), SALK_073818C (At5g11670), CS83358. 5 (At5g11670) and SALK_139336C (At5g25880), the six mutants were tested. The results showed that the salt tolerance of the two mutants of SALK_064163 and SALK_073818C was significantly lower than that of the wild type. The germination rate, germination potential and the length of the main root of the two mutant lines of SALK_064163 and SALK_073818C were significantly lower than those of the wild type under salt stress. Arabidopsis, the two salt sensitive mutants were selected for follow-up experiments. In the seedling stage, the activity of Pn, Ci, Tr and Gs of SALK_064163 and SALK_073818C were significantly lower than those of wild type Arabidopsis under 100 mM NaCl treatments. The content of chlorophyll, the diameter of PSII, Fv/Fm and delta I/Io were significantly lower than those of the wild type. The plant, while Fo, 1-qp and NPQ were significantly higher than the wild type, and the fresh weight and dry weight of the two mutants were significantly lower than those of the wild type. These results showed that under salt stress, the light system of SALK_064163 and SALK_073818C was higher than the wild type, and its salt tolerance was significantly lower than that of the wild type. The results indicated that the sweet sorghum NADP-ME was significantly lower than that of the wild type. Genes can enhance photosynthetic capacity of Arabidopsis seedlings under salt stress.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S566.5;Q943.2
本文编号：2167996
[Abstract]:By analyzing the results of the transcriptional sequence, we speculated that the NADP-ME gene was related to the salt tolerance, and then extracted the NADP-ME gene from the sweet sorghum leaves and transferred to Arabidopsis and carried out the expression and functional analysis of the gene. The main results were as follows: 1, in order to study the salt tolerance mechanism of sweet sorghum, the sweet sorghum seedlings were treated with 0 mM and 150 mM NaCl. The KEGG pathway analysis showed that 150 genes were annotated to 70 pathways in M-81E, and there were 174 gene annotations to 63 pathways in Rome and the differential expression genes under salt stress in 63 pathways, mainly concentrated in the photosynthetic carbon fixation pathway, and the NADP-ME gene was annotated to photosynthesis. Under salt stress, the gene expression in M-81E is quite high, but there is no obvious change in Rome. We speculate that NADP-ME may affect the salt tolerance of sweet sorghum by regulating photosynthetic efficiency, so NADP-ME gene is isolated from sweet sorghum, and its function.2 is verified. We set up a known NADP-ME sequence based on sweet sorghum. Primers. The NADP-ME gene was cloned from sweet sorghum leaves by PCR. The gene was 1911 BP and 637 amino acids were encoded. The molecular weight was about 15.7 kDa.. The homology of the NADP-ME gene in sweet sorghum leaves was found to be the highest, reaching 98% and 96%.3. The NADP-ME gene and pCAMBIA3300 vector were added to the sweet sorghum leaves. The overexpressed plant was infected by the method of inflorescence infection. The overexpressed plant was screened by Basta screening marker and the overexpressed strain.4 was detected at the level of DNA. Under salt stress, the germination rate, germination potential and the length of the main root of the wild Arabidopsis and overexpressed lines were suppressed, but the wild type was inhibited. The test results showed that the salt tolerance of the overexpressed strain was better in the germination period, and the expression amount of the sweet sorghum NADP-ME gene under the different salt stress was detected by RT-PCR. The results showed that the expression of the gene was the highest under the treatment of 100 mM NaCl. Therefore, 0 mM and 100 mM NaCl were selected for the treatment of Arabidopsis plants. Test the photosynthetic parameters, chlorophyll content, fluorescence parameters and the activity of I in the light system. The results showed that the photosynthetic activity of overexpressed lines was higher than that of wild type without salt treatment, and the other physiological indexes were basically the same as those of wild type. Under salt stress, the content of Pn, Ci, Tr and Gs in overexpressed lines was significantly higher than that of wild Arabidopsis. The expression of this gene could improve the photosynthetic efficiency of Arabidopsis under salt stress. The content of chlorophyll, PSII, Fv/Fm and delta I/Io of overexpressed lines were significantly higher than that of wild Arabidopsis, and Fo, 1-qp and NPQ were significantly lower than those of wild Arabidopsis. These results indicated that the photosynthetic capacity of overexpressed lines was stronger under salt stress. We also studied the salt tolerance of the plant lines. Under salt stress, the fresh weight and dry weight of overexpressed lines were significantly higher than those of the wild type. It showed that the salt tolerance of overexpressed lines was higher than that of wild type.5, SALK_064163 (At1g79750), CS855818 (At1g79750), SALK_036898 (At2g19900), SALK_073818C (At5g11670), CS83358. 5 (At5g11670) and SALK_139336C (At5g25880), the six mutants were tested. The results showed that the salt tolerance of the two mutants of SALK_064163 and SALK_073818C was significantly lower than that of the wild type. The germination rate, germination potential and the length of the main root of the two mutant lines of SALK_064163 and SALK_073818C were significantly lower than those of the wild type under salt stress. Arabidopsis, the two salt sensitive mutants were selected for follow-up experiments. In the seedling stage, the activity of Pn, Ci, Tr and Gs of SALK_064163 and SALK_073818C were significantly lower than those of wild type Arabidopsis under 100 mM NaCl treatments. The content of chlorophyll, the diameter of PSII, Fv/Fm and delta I/Io were significantly lower than those of the wild type. The plant, while Fo, 1-qp and NPQ were significantly higher than the wild type, and the fresh weight and dry weight of the two mutants were significantly lower than those of the wild type. These results showed that under salt stress, the light system of SALK_064163 and SALK_073818C was higher than the wild type, and its salt tolerance was significantly lower than that of the wild type. The results indicated that the sweet sorghum NADP-ME was significantly lower than that of the wild type. Genes can enhance photosynthetic capacity of Arabidopsis seedlings under salt stress.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S566.5;Q943.2
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