發(fā)布時(shí)間：2018-11-16 12:18

【摘要】：小麥?zhǔn)侨祟愔匾募Z食資源,干旱是導(dǎo)致小麥產(chǎn)量降低的重要原因之一。LEA蛋白作為一種重要的抗逆蛋白,在植物遭受外界不良條件時(shí)大量表達(dá),可以減輕植物細(xì)胞受害,發(fā)揮對植物的保護(hù)功能。LEA3蛋白是LEA蛋白家族的重要成員,具有高度親水性,已有實(shí)驗(yàn)證明該族LEA蛋白與植物抗旱能力相關(guān)。本文以實(shí)驗(yàn)室已經(jīng)克隆得到的“鄭引1號”小麥中的lea3基因TaDRLea3-2為基礎(chǔ),通過轉(zhuǎn)化原核生物大腸桿菌和擬南芥,研究小麥TaDRLea3-2蛋白在非生物脅迫下對大腸桿菌和擬南芥抗逆能力的影響。并通過體外酶活實(shí)驗(yàn)研究在高溫和冷凍處理下TaDRLea3-2蛋白對LDH酶活的保護(hù)作用。為進(jìn)一步了解LEA蛋白在抵抗非生物脅迫中的功能和作用機(jī)制奠定了基礎(chǔ)。通過研究獲得以下結(jié)果:1.在已經(jīng)克隆得到小麥TaDRLea3-2基因的基礎(chǔ)上,成功構(gòu)建了原核表達(dá)載體pET28a-TaDRLea3-2,熱激法轉(zhuǎn)化大腸桿菌BL21(DE3)。SDS-PAGE檢測大腸桿菌表達(dá)的重組蛋白的分子量在23 kD附近。用組氨酸抗體對純化后的蛋白進(jìn)Weatern Blot鑒定,表明得到的蛋白為TaDRLea3-2蛋白。2.體外酶活保護(hù)實(shí)驗(yàn)表明TaDRLea3-2蛋白可以保護(hù)LDH酶在液氮凍融處理和45℃高溫處理下酶活性。3.通過對含有空載的對照菌BL21-DE3(pET28a)和含有目的基因的重組菌BL21-DE3(pET28a-TaDRLea3-2)進(jìn)行高溫,低溫,高鹽和高滲處理,表達(dá)TaDRLea3-2蛋白的大腸桿菌對這些非生物脅迫的耐受性較對照菌增加。4.通過農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化,將構(gòu)建成功的植物表達(dá)載體pBI121-TaDRLea3-2轉(zhuǎn)化野生型擬南芥,對轉(zhuǎn)基因擬南芥篩選和鑒定,成功得到T3代轉(zhuǎn)基因擬南芥的種子。以野生型和T3轉(zhuǎn)基因擬南芥的種子為材料進(jìn)行抗旱性研究。在幼苗期,在濃度為150 mM、200 mM、250 mM甘露醇處理下,轉(zhuǎn)基因擬南芥的根長大于野生型擬南芥。在成苗期,通過自然干旱處理,轉(zhuǎn)基因株系表現(xiàn)出更高的葉片含水量和葉綠素含量,低的丙二醛含量和高的脯氨酸的積累量。表明小麥TaDRLea3-2基因在擬南芥中過表達(dá)提高了轉(zhuǎn)基因擬南芥的抗旱性。
[Abstract]:Wheat is an important food resource for human being. Drought is one of the important reasons leading to the decrease of wheat yield. As an important stress resistance protein, LEA protein can be expressed in large quantities when plants suffer from external adverse conditions, which can alleviate the damage of plant cells. LEA3 protein is an important member of LEA protein family and has high hydrophilicity. It has been proved that the LEA protein of this family is related to plant drought resistance. Based on the lea3 gene TaDRLea3-2 of "Zhengyin 1" wheat which has been cloned in the laboratory, the transformation of Escherichia coli and Arabidopsis thaliana was carried out by the transformation of E. coli and Arabidopsis thaliana. To study the effect of wheat TaDRLea3-2 protein on stress resistance of Escherichia coli and Arabidopsis thaliana under abiotic stress. The protective effect of TaDRLea3-2 protein on LDH activity was studied by enzyme activity in vitro. It lays a foundation for further understanding the function and mechanism of LEA protein in resisting abiotic stress. The results are as follows: 1. Based on the cloning of wheat TaDRLea3-2 gene, the prokaryotic expression vector pET28a-TaDRLea3-2, heat shock method was successfully constructed to transform Escherichia coli BL21 (DE3). The molecular weight of the recombinant protein detected by SDS-PAGE was around 23 kD. The purified protein was identified by Weatern Blot with histidine antibody, and the protein was identified as TaDRLea3-2 protein. 2. 2. In vitro enzyme protection experiments showed that TaDRLea3-2 protein could protect the enzyme activity of LDH under liquid nitrogen freeze-thaw treatment and 45 鈩，

本文編號：2335503