鹽脅迫下甜高粱NADP-ME基因的功能分析
發(fā)布時間：2018-08-06 14:17
【摘要】：本試驗通過分析轉錄組測序結果推測NADP-ME基因與耐鹽性相關,進而從甜高粱葉片中提取NADP-ME基因,轉入擬南芥并進行該基因的表達和功能分析。主要試驗結果如下:1、為了研究甜高粱的耐鹽機理,我們用0 mM和150 mM NaCl處理甜高粱幼苗,提取葉片RNA進行轉錄組測序。通過KEGG途徑分析發(fā)現(xiàn),在M-81E中有150個基因注釋到70個通路中,而在羅馬中有174個基因注釋到63個通路中。M-81E和羅馬在鹽脅迫下的差異表達基因,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中的表達量相當高,然而在羅馬中卻沒有明顯變化。我們推測NADP-ME可能通過調節(jié)光合效率來影響甜高粱耐鹽性,所以從甜高粱中分離了NADP-ME基因,并驗證其功能。2、我們根據甜高粱已知NADP-ME序列設計引物。通過PCR的方法從甜高粱葉片中克隆NADP-ME基因,該基因全長1911 bp,編碼637個氨基酸,分子質量約為15.7 kDa。經過同源序列比對發(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,達到98%和96%。3、將NADP-ME基因與pCAMBIA3300載體進行重組,利用花序侵染的方法侵染擬南芥,獲得過表達植株。通過Basta篩選標記對過表達株系進行篩選,在DNA水平檢測過表達株系。4、在鹽脅迫下,野生型擬南芥與過表達株系的萌發(fā)率、萌發(fā)勢和主根長度都受到了抑制,但野生型受到的抑制程度較嚴重,從試驗結果可以看出,在萌發(fā)期過表達株系的耐鹽能力較好;用RT-PCR檢測過表達株系中甜高粱NADP-ME基因在不同鹽脅迫下的表達量,結果發(fā)現(xiàn)在100 mM NaCl處理下該基因的表達量最高。因此選用0 mM和100 mM NaCl處理擬南芥植株,測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。結果表明,未經鹽處理條件下,過表達株系的光合活性比野生型高,其它生理指標與野生型基本一致。在鹽脅迫下,過表達株系的Pn、Ci、Tr以及Gs的含量顯著高于野生型擬南芥,說明該基因的表達能夠提高擬南芥在鹽脅迫下的光合效率。過表達株系的葉綠素含量、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。這些結果說明在鹽脅迫下,過表達株系的光合能力較強。為了進一步研究過表株系的耐鹽性,我們又對其生物量進行了測定,在鹽脅迫下,過表達株系的鮮重和干重都明顯高于野生型。說明過表達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),這六個突變體進行試驗,結果發(fā)現(xiàn),SALK_064163和SALK_073818C這兩個突變體的耐鹽能力顯著低于野生型。鹽脅迫下,SALK_064163和SALK_073818C這兩個突變體株系在萌發(fā)期的萌發(fā)率、萌發(fā)勢和主根長度明顯低于野生型擬南芥,因此選用了這兩個鹽敏感突變體進行后續(xù)試驗。在幼苗期,通過測定各項生理指標發(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,并且這兩個突變體的鮮重和干重都明顯低于野生型。這些結果表明,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.
【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S566.5;Q943.2
本文編號：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.
【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S566.5;Q943.2
【參考文獻】
相關期刊論文 前10條
1 張華鋒;馬峰;張懷友;王明友;;鹽脅迫對西葫蘆葉片光合生理特性的影響[J];現(xiàn)代農業(yè)科技;2015年16期
2 徐煥文;劉宇;姜靜;劉桂豐;;鹽脅迫對白樺光合特性及葉綠素熒光參數(shù)的影響[J];西南林業(yè)大學學報;2015年04期
3 曹冰;龍翔宇;肖小虎;唐朝榮;;巴西橡膠樹中2個NADP-蘋果酸酶基因的克隆和表達特性分析[J];熱帶作物學報;2014年05期
4 劉曉龍;徐晨;徐克章;崔菁菁;安久海;凌鳳樓;張治安;武志海;;鹽脅迫對水稻葉片光合作用和葉綠素熒光特性的影響[J];作物雜志;2014年02期
5 董慶龍;王海榮;安淼;余賢美;王長君;;蘋果NADP依賴的蘋果酸酶基因克隆、序列和表達分析[J];中國農業(yè)科學;2013年09期
6 魯紅俠;楊艷;朱小茜;;NaCl脅迫對擬南芥種子萌發(fā)與幼苗生長的影響[J];安徽農業(yè)科學;2013年08期
7 陸玉建;高春明;鄭香峰;鈕松召;;鹽脅迫對擬南芥種子萌發(fā)的影響[J];湖北農業(yè)科學;2012年22期
8 楊景寧;王彥榮;;NaCl脅迫對四種荒漠植物種子萌發(fā)的影響[J];草業(yè)學報;2012年05期
9 李秀峰;張欣欣;高野哲夫;柳參奎;;水稻(Oryza sativa L.)蘋果酸酶(OsNADP-ME3)基因在逆境下的表達特性研究[J];基因組學與應用生物學;2012年04期
10 張嵐;李慶章;;新基因功能研究的整體策略[J];中國畜牧獸醫(yī);2011年05期
相關碩士學位論文 前4條
1 杜西河;玉米PEPC，，PPDK和NADP-ME基因在擬南芥中的表達分析[D];河南農業(yè)大學;2013年
2 程歡;鹽脅迫對擬南芥生長影響的生理生化及根的蛋白組學研究[D];湖北大學;2012年
3 劉暢;水稻NADP-ME同工酶在逆境環(huán)境下的功能差異分析[D];東北林業(yè)大學;2011年
4 郎志紅;鹽堿脅迫對植物種子萌發(fā)和幼苗生長的影響[D];蘭州交通大學;2008年
本文編號：2167996
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2167996.html
最近更新
教材專著
