本文選題：馬鈴薯　切入點(diǎn)：MAPKK基因　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：MAPKKs(即絲裂原活化蛋白激酶激酶)是主要存在于動(dòng)植物細(xì)胞中,是一種信號(hào)傳遞者,在細(xì)胞中具有重要作用,而且也是MAPK級(jí)聯(lián)反應(yīng)中的關(guān)鍵組成部分。MAPK信號(hào)通路在動(dòng)植物細(xì)胞中具有明顯的級(jí)聯(lián)現(xiàn)象,是信號(hào)轉(zhuǎn)導(dǎo)途徑主要途徑之一,并在植物體內(nèi)起到關(guān)鍵作用,它能被激素、細(xì)胞因子等不同的細(xì)胞外物質(zhì)刺激,從而使細(xì)胞在生長(zhǎng)、發(fā)育等多種重要的生理過程發(fā)生一列反應(yīng)來響應(yīng)外界刺激脅迫。MAPK信號(hào)通路在所有的動(dòng)物、植物、微生物等真核生物中廣泛存在。迄今為止,MAPK級(jí)聯(lián)反應(yīng)只在有限的植物,如擬南芥、水稻和楊樹等物種中有過相關(guān)的系統(tǒng)研究,而MAPKK家族基因在馬鈴薯中的系統(tǒng)研究還沒有相關(guān)的文獻(xiàn)報(bào)導(dǎo)。本研究通過生物信息學(xué)分析了馬鈴薯MAPKK基因,鑒定了5個(gè)MAPKK家族基因。通過對(duì)馬鈴薯模擬各種生物脅迫和非生物脅迫,篩選出了與干旱相關(guān)的MAPKK基因。構(gòu)建了MAPKK1基因的過表達(dá)載體和Gateway-MAPKK1的干擾表達(dá)載體,轉(zhuǎn)化了馬鈴薯“大西洋”和“紫花白”兩個(gè)品種獲得了轉(zhuǎn)基因馬鈴薯植株。用qRT-PCR分析了轉(zhuǎn)基因植株中MAPKK1基因的表達(dá)量變化。取得的主要研究成果如下:1.通過生物信息學(xué)分析,鑒定出5個(gè)馬鈴薯St MAPKK基因�？煞譃锳、B、C、D四組,其中馬鈴薯StMAPKK基因全長(zhǎng)cDNA的平均長(zhǎng)度為1001 bp,從1547bp至1574bp不等。馬鈴薯StMAPKKs編碼的氨基酸序列長(zhǎng)度從333個(gè)氨基酸至515個(gè)氨基酸之間,StMAPKKs的預(yù)測(cè)蛋白質(zhì)的分子量變化從37.26 kDa至57.45 kDa。推導(dǎo)的多肽的等電點(diǎn)(PI)是從5.47至8.87。而染色體則主要集中于12號(hào)染色體和3號(hào)染色體。2.通過qRT-PCR分析了馬鈴薯在生物脅迫和非生物脅迫處理下(4℃、45℃、20%PEG、200 mM NaCl、10 mM H2O2、100μM MeJA、100μM SA、100μM ABA)馬鈴薯StMAPKK家族基因表達(dá)量的變化。以及干旱脅迫下馬鈴薯StMAPKK基因的表達(dá)模式,結(jié)果表明在干旱脅迫下MAPKK中多數(shù)基因均上調(diào)表達(dá),其中StMAPKK1、StMAPKK5上調(diào)程度最高。3.選取干旱脅迫下表達(dá)量上調(diào)最明顯的基因MAPKK1用于構(gòu)建過表達(dá)載體。成功構(gòu)建與抗旱相關(guān)的MAPKK1過表達(dá)載體,經(jīng)農(nóng)桿菌轉(zhuǎn)化后,分別進(jìn)行PCR鑒定并成功獲得相應(yīng)的轉(zhuǎn)基因StMAPKK1轉(zhuǎn)化植株。經(jīng)qRT-PCR檢測(cè),其轉(zhuǎn)基因馬鈴薯“大西洋”品種中,StMAPKK1表達(dá)量是對(duì)照的3.2倍;而轉(zhuǎn)基因馬鈴薯“紫花白”品種中,StMAPKK1表達(dá)量最高為對(duì)照“紫花白”品種的7.2倍。4.用Gateway技術(shù)構(gòu)建MAPKK1基因干擾表達(dá)載體。經(jīng)農(nóng)桿菌遺傳轉(zhuǎn)化后,分別進(jìn)行PCR鑒定并成功獲得相應(yīng)的轉(zhuǎn)基因轉(zhuǎn)化植株。經(jīng)qRT-PCR檢測(cè),其轉(zhuǎn)基因馬鈴薯“大西洋”品種中,StMAPKK1表達(dá)量最低是對(duì)照“大西洋”品種的0.09倍;而轉(zhuǎn)基因馬鈴薯“紫花白”品種中,StMAPKK1表達(dá)量最低為對(duì)照“紫花白”品種的0.4倍。StMAPKK1基因表達(dá)量均得到明顯的抑制作用。
[Abstract]:MAPKKs (mitogen-activated protein kinase kinase) is mainly found in animal and plant cells, is a signal transporter, and plays an important role in cells.It is also a key component of MAPK cascade reaction. MAPK signaling pathway has obvious cascade phenomenon in animal and plant cells, is one of the main pathways of signal transduction, and plays a key role in plants, it can be hormone,Different extracellular substances such as cytokines stimulate cells in many important physiological processes, such as growth and development, to respond to external stimuli. MAPK signaling pathways are present in all animals and plants.Eukaryotes such as microbes are widespread.Up to now, the cascade reaction of mitogen-activated kinase (MAPK) has been systematically studied in a limited number of plants, such as Arabidopsis thaliana, rice and poplar, but the systematic study of MAPKK family genes in potato has not been reported.In this study, the MAPKK gene of potato was analyzed by bioinformatics, and five MAPKK family genes were identified.The MAPKK genes related to drought were screened by simulating various biotic and abiotic stresses on potato.The overexpression vector of MAPKK1 gene and the interference expression vector of Gateway-MAPKK1 were constructed and transformed into potato "Atlantic" and "purple flower white" to obtain transgenic potato plants.The changes of MAPKK1 gene expression in transgenic plants were analyzed by qRT-PCR.The main research results are as follows: 1.Five St MAPKK genes were identified by bioinformatics analysis.The average length of full-length cDNA of potato StMAPKK gene was 1001 BP, ranging from 1547bp to 1574bp.The predicted molecular weight of the predicted protein of potato StMAPKKs encoded amino acid sequence ranged from 333 amino acids to 515 amino acids from 37.26 kDa to 57.45 kDa.The isoelectric point (Pi) of the deduced polypeptide is from 5.47 to 8.87.The chromosomes were mainly concentrated on chromosome 12 and chromosome 2. 2.The changes of StMAPKK gene expression in potato StMAPKK family under biological stress and abiotic stress were analyzed by qRT-PCR.And the expression pattern of StMAPKK gene in potato under drought stress. The results showed that most of the genes were up-regulated in MAPKK under drought stress, among which StMAPK1 + StMAPKK5 was the highest.The gene MAPKK1, which was the most obvious up-regulation gene under drought stress, was used to construct overexpression vector.The MAPKK1 overexpression vector related to drought resistance was successfully constructed. After transformed by Agrobacterium tumefaciens, the transformed plants were identified by PCR and the corresponding transgenic StMAPKK1 plants were obtained successfully.QRT-PCR analysis showed that the expression of StMAPKK1 in the transgenic potato "Atlantic" was 3.2 times higher than that in the control, while the highest expression of StMAPKK1 in the transgenic potato "purple flower white" was 7.2 times as high as that in the control.The interference expression vector of MAPKK1 gene was constructed by Gateway technique.After genetic transformation of Agrobacterium tumefaciens, the transgenic plants were identified by PCR and the corresponding transgenic plants were obtained successfully.QRT-PCR analysis showed that the expression of StMAPKK1 in transgenic potato "Atlantic" was 0.09 times of that of the control.However, the expression of StMAPKK1 in transgenic potato "Zihuabai" was the lowest, which was 0.4 times as much as that of "Zihuabai", and the expression of StMAPKK1 was significantly inhibited.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S532

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