本文選題：檉柳 + ThZFP3　； 參考：《東北林業(yè)大學(xué)》2016年碩士論文

【摘要】：ZFP (Zinc Finger Proteins)轉(zhuǎn)錄因子是植物中的一大類重要轉(zhuǎn)錄調(diào)控子,主要在植物生長(zhǎng)發(fā)育、抗逆境脅迫等方面具有重要的作用。本研究從檉柳轉(zhuǎn)錄組數(shù)據(jù)中鑒定出一條鹽脅迫響應(yīng)的鋅指蛋白基因,命名為ThZFP3,該基因全長(zhǎng)1335 bp,編碼444氨基酸,屬于CCCH型鋅指蛋白基因家族,編碼蛋白相對(duì)分子質(zhì)量48 491.6 Da,理論等電點(diǎn)(PI)為8.08。構(gòu)建植物表達(dá)載體pBI121-GFP-ThZFP3,利用基因槍技術(shù)瞬時(shí)轉(zhuǎn)化洋蔥表皮研究亞細(xì)胞定位,結(jié)果表明ThZFP3蛋白定位在細(xì)胞核中。利用酵母雙雜交技術(shù)鑒定ThZFP3轉(zhuǎn)錄激活結(jié)構(gòu)域,確定其轉(zhuǎn)錄激活區(qū)位于C端的225～280氨基酸區(qū)段。利用實(shí)時(shí)熒光定量RT-PCR技術(shù)分析顯示,該基因受高鹽、干旱誘導(dǎo)上調(diào)表達(dá),暗示ThZFP3可能在檉柳抗旱耐鹽過程中發(fā)揮重要作用。構(gòu)建ThZFP3基因的過表達(dá)載體,轉(zhuǎn)化擬南芥,獲得T3代轉(zhuǎn)基因株系。對(duì)野生型和轉(zhuǎn)基因株系擬南芥進(jìn)行NaCl和Mannitol脅迫處理,脅迫條件下轉(zhuǎn)基因株系的萌發(fā)率、根長(zhǎng)、鮮重及長(zhǎng)勢(shì)都優(yōu)于野生型株系,表明ThZFP3基因能提高轉(zhuǎn)基因擬南芥的耐鹽性和耐旱性。構(gòu)建RNAi載體pC5941-ThZFP3,利用瞬時(shí)轉(zhuǎn)化技術(shù)將農(nóng)桿菌pROKⅡ-ThZFP3、 pC5941-ThZFP3和pROKⅡ轉(zhuǎn)入檉柳,獲得過表達(dá)(OE)、抑制表達(dá)(RNAi)和對(duì)照(Control)轉(zhuǎn)基因植株,分析三種轉(zhuǎn)基因植株在NaCl和Mannitol脅迫下的抗逆能力。DAB、NBT和Evans blue組織化學(xué)染色結(jié)果顯示,過表達(dá)植株(OE)細(xì)胞內(nèi)的02-和1-1202含量明顯低于比Control和RNAi植株,說明過表達(dá)株系受脅迫后細(xì)胞膜結(jié)構(gòu)較為完整,受損傷程度低,而抑制表達(dá)株系細(xì)胞受損傷程度最嚴(yán)重。測(cè)定三種轉(zhuǎn)基因植株的失水率、葉綠素含量、PRO含量、H202含量、MDA含量、POD和SOD活性等生理指標(biāo),結(jié)果顯示過表達(dá)植株(OE)的失水率、ROS含量和MDA含量相對(duì)最低,葉綠素含量、PRO含量、POD活性和SOD活性相對(duì)最高;RNAi植株的失水率、ROS含量和MDA含量最高,葉綠素含量、PRO含量、POD和SOD活性相對(duì)最低。以上研究表明,ThZFP3能夠提高轉(zhuǎn)基因植株活性氧(ROS)清除能力,降低細(xì)胞受損傷程度,從而提高植株的抗逆能力。
[Abstract]:ZFP Zinc Finger proteins is a kind of important transcription regulators in plants, which plays an important role in plant growth and development, stress resistance and so on. In this study, a zinc finger protein gene, named ThZFP3, was identified from Tamarix chinensis transcriptional data. The gene is 1335 BP in length and encodes 444-amino acid. It belongs to the CCCH type zinc finger protein gene family. The relative molecular weight of the encoded protein is 48 491.6 Da, and the theoretical isoelectric point (Pi) is 8.08. The plant expression vector pBI121-GFP-ThZFP3 was constructed, and the subcellular localization of onion epidermis was studied by gene gun technique. The results showed that the ThZFP3 protein was located in the nucleus. Yeast two-hybrid technique was used to identify the transcriptional activation domain of ThZFP3. Real-time fluorescence quantitative RT-PCR analysis showed that the gene was overexpressed by high salt and up-regulated by drought, suggesting that ThZFP3 might play an important role in the process of drought and salt tolerance in Tamarix chinensis. Overexpression vector of ThZFP3 gene was constructed and transformed into Arabidopsis thaliana. Under the stress of NaCl and Mannitol, the germination rate, root length, fresh weight and growth of transgenic Arabidopsis were better than those of wild type. The results showed that ThZFP3 gene could improve salt tolerance and drought tolerance of transgenic Arabidopsis thaliana. RNAi vector pC5941-ThZFP3 was constructed. Agrobacterium tumefaciens pROK 鈪，

本文編號(hào)：1978056