鹽脅迫下甜高粱NADP-ME基因的功能分析
發(fā)布時(shí)間：2018-08-06 14:17
【摘要】：本試驗(yàn)通過(guò)分析轉(zhuǎn)錄組測(cè)序結(jié)果推測(cè)NADP-ME基因與耐鹽性相關(guān),進(jìn)而從甜高粱葉片中提取NADP-ME基因,轉(zhuǎn)入擬南芥并進(jìn)行該基因的表達(dá)和功能分析。主要試驗(yàn)結(jié)果如下:1、為了研究甜高粱的耐鹽機(jī)理,我們用0 mM和150 mM NaCl處理甜高粱幼苗,提取葉片RNA進(jìn)行轉(zhuǎn)錄組測(cè)序。通過(guò)KEGG途徑分析發(fā)現(xiàn),在M-81E中有150個(gè)基因注釋到70個(gè)通路中,而在羅馬中有174個(gè)基因注釋到63個(gè)通路中。M-81E和羅馬在鹽脅迫下的差異表達(dá)基因,主要集中在光合碳固定途徑中,并且NADP-ME基因被注釋到光合作用途徑中。在鹽脅迫下,該基因在M-81E中的表達(dá)量相當(dāng)高,然而在羅馬中卻沒(méi)有明顯變化。我們推測(cè)NADP-ME可能通過(guò)調(diào)節(jié)光合效率來(lái)影響甜高粱耐鹽性,所以從甜高粱中分離了NADP-ME基因,并驗(yàn)證其功能。2、我們根據(jù)甜高粱已知NADP-ME序列設(shè)計(jì)引物。通過(guò)PCR的方法從甜高粱葉片中克隆NADP-ME基因,該基因全長(zhǎng)1911 bp,編碼637個(gè)氨基酸,分子質(zhì)量約為15.7 kDa。經(jīng)過(guò)同源序列比對(duì)發(fā)現(xiàn),甜高粱葉片中NADP-ME基因與白茅和玉米的同源性最高,達(dá)到98%和96%。3、將NADP-ME基因與pCAMBIA3300載體進(jìn)行重組,利用花序侵染的方法侵染擬南芥,獲得過(guò)表達(dá)植株。通過(guò)Basta篩選標(biāo)記對(duì)過(guò)表達(dá)株系進(jìn)行篩選,在DNA水平檢測(cè)過(guò)表達(dá)株系。4、在鹽脅迫下,野生型擬南芥與過(guò)表達(dá)株系的萌發(fā)率、萌發(fā)勢(shì)和主根長(zhǎng)度都受到了抑制,但野生型受到的抑制程度較嚴(yán)重,從試驗(yàn)結(jié)果可以看出,在萌發(fā)期過(guò)表達(dá)株系的耐鹽能力較好;用RT-PCR檢測(cè)過(guò)表達(dá)株系中甜高粱NADP-ME基因在不同鹽脅迫下的表達(dá)量,結(jié)果發(fā)現(xiàn)在100 mM NaCl處理下該基因的表達(dá)量最高。因此選用0 mM和100 mM NaCl處理擬南芥植株,測(cè)定葉片中的光合參數(shù)、葉綠素含量、熒光參數(shù)和光系統(tǒng)I的活性。結(jié)果表明,未經(jīng)鹽處理?xiàng)l件下,過(guò)表達(dá)株系的光合活性比野生型高,其它生理指標(biāo)與野生型基本一致。在鹽脅迫下,過(guò)表達(dá)株系的Pn、Ci、Tr以及Gs的含量顯著高于野生型擬南芥,說(shuō)明該基因的表達(dá)能夠提高擬南芥在鹽脅迫下的光合效率。過(guò)表達(dá)株系的葉綠素含量、ΦPSII、Fv/Fm及ΔI/Io活性顯著高于野生型擬南芥,Fo、1-qp和NPQ顯著低于野生型擬南芥。這些結(jié)果說(shuō)明在鹽脅迫下,過(guò)表達(dá)株系的光合能力較強(qiáng)。為了進(jìn)一步研究過(guò)表株系的耐鹽性,我們又對(duì)其生物量進(jìn)行了測(cè)定,在鹽脅迫下,過(guò)表達(dá)株系的鮮重和干重都明顯高于野生型。說(shuō)明過(guò)表達(dá)株系耐鹽程度高于野生型。5、選取SALK_064163(At1g79750),CS855818(At1g79750),SALK_036898(At2g19900),SALK_073818C(At5g11670),CS833585(At5g11670)和SALK_139336C(At5g25880),這六個(gè)突變體進(jìn)行試驗(yàn),結(jié)果發(fā)現(xiàn),SALK_064163和SALK_073818C這兩個(gè)突變體的耐鹽能力顯著低于野生型。鹽脅迫下,SALK_064163和SALK_073818C這兩個(gè)突變體株系在萌發(fā)期的萌發(fā)率、萌發(fā)勢(shì)和主根長(zhǎng)度明顯低于野生型擬南芥,因此選用了這兩個(gè)鹽敏感突變體進(jìn)行后續(xù)試驗(yàn)。在幼苗期,通過(guò)測(cè)定各項(xiàng)生理指標(biāo)發(fā)現(xiàn),在100 mM NaCl處理下,SALK_064163和SALK_073818C的Pn、Ci、Tr和Gs的活性都顯著低于野生型擬南芥,葉綠素含量、ΦPSII、Fv/Fm及ΔI/Io活性均顯著低于野生型植株,而Fo、1-qp和NPQ顯著高于野生型植株,并且這兩個(gè)突變體的鮮重和干重都明顯低于野生型。這些結(jié)果表明,在鹽脅迫下,SALK_064163和SALK_073818C的光系統(tǒng)受鹽害程度高于野生型,并且其耐鹽性明顯低于野生型。上述結(jié)果表明,甜高粱NADP-ME基因能夠提高擬南芥幼苗在鹽脅迫下的光合能力。
[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.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S566.5;Q943.2
本文編號(hào)：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.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S566.5;Q943.2
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