本文選題：皺波角叉菜　切入點：CaM　出處：《遼寧師范大學》2016年碩士論文

【摘要】：植物生長環(huán)境的變化會影響植物的生長與代謝。藻類應答環(huán)境脅迫的信號通路及相應蛋白質功能的研究已成為熱點。作為機體受到一系列如高溫、干旱和嚴寒等非生物脅迫后大量表達的應激蛋白,HSP70(Heat Shock protein 70)廣泛參與生物體內(nèi)變性蛋白質的復性和降解,對機體的代謝調控和響應環(huán)境脅迫具有重要的作用。隨著溫室效應的加劇,災害性高溫氣候成為影響藻類生長的一種主要的非生物脅迫。面對高溫脅迫,植物體會快速啟動復雜的調控機制來應對熱刺激,鈣-鈣調素熱激信號轉導通路作為響應熱脅迫主要的調控機制,可以調節(jié)HSP70的表達。本實驗以經(jīng)濟海藻皺波角叉菜的葉狀體為實驗材料,運用RT-PCR技術克隆皺波角叉菜的CcCaM、CcHSP70基因的ORF序列。利用生物軟件對序列進行分析,結果顯示CcCaM基因的ORF全長為453bp,共編碼150個氨基酸;CcHSP70基因的ORF全長為1971bp,共編碼656個氨基酸,并預測出在CcHSP70的氨基酸序列中包含一段由21個氨基酸殘基組成的CaM結合區(qū)域,這表明在皺波角叉菜中可能存在著通過CaM與HSP70相結合進而來調節(jié)熱激信號轉導的下游事件,這為進一步研究皺波角叉菜的熱激信號轉導通路提供依據(jù)。為了探討HSP70在皺波角叉菜抵抗高溫與冷脅迫中的作用及響應特點,本研究采用Real-Time PCR技術檢測皺波角叉菜葉狀體在28℃高溫脅迫與4℃冷脅迫下CcHSP70基因相對表達量的變化。并將CcHSP70基因轉入大腸桿菌內(nèi)進行原核表達與分離純化,隨后檢測了CcHSP70蛋白質的ATPase活性,測定45℃高溫與10℃冷脅迫下轉pET28a-CcHSP70大腸桿菌的生長情況。結果顯示:CcHSP70基因的表達受到高溫脅迫與冷脅迫的顯著誘導,在持續(xù)28℃高溫脅迫下,CcHSP70的表達量在4 h時達到峰值,之后一直保持在較高的水平與對照差異顯著。在持續(xù)冷脅迫下,在8 h時CcHSP70表達量與對照組相比達到顯著差異。CcHSP70具有ATPase活性,其可通過ATP的水解循環(huán)來控制多肽的結合和釋放。在45℃高溫與10℃冷脅迫下CcHSP70的過表達提高了大腸桿菌的抗高溫能力與耐冷性。結果表明CcHSP70受熱激與冷脅迫的誘導表達,在高溫脅迫與冷脅迫中維持了細胞蛋白的穩(wěn)定,對細胞具有重要的保護作用。
[Abstract]:The changes of plant growth environment will affect the growth and metabolism of plants. The research on the signal pathway of algae response to environmental stress and the corresponding protein function has become a hotspot. As a series of organisms subject to a series of high temperatures, The stress protein (HSP70 Heat Shock protein 70), which is widely expressed after abiotic stress such as drought and cold, is widely involved in the renaturation and degradation of denatured proteins in organisms. With the aggravation of Greenhouse Effect, catastrophic high temperature climate has become a major abiotic stress affecting algae growth. Plants experience the rapid initiation of complex regulatory mechanisms to respond to heat stimulation, calcium calmodulin heat shock signal transduction pathway as the main regulatory mechanism in response to heat stress, The RT-PCR technique was used to clone the ORF sequence of the CcCaMU CcHSP70 gene from the striatum of economic algal carrageenia crassifolia, and the sequence was analyzed by biological software. The results showed that the total length of ORF of CcCaM gene was 453bpand the total length of ORF encoding 150 amino acids CcHSP70 gene was 1971bp. the total length of ORF encoding 656 amino acids was predicted to include a CaM binding region composed of 21 amino acid residues in the amino acid sequence of CcHSP70. This suggests that there may be downstream events that regulate heat shock signal transduction through the combination of CaM and HSP70 in Carrageenia crassifolia. This provides a basis for further study on the heat shock signal transduction pathway of carrageenia crassifolia. In order to investigate the role and response of HSP70 in the resistance to high temperature and cold stress of Carrageenia crassifolia, In this study, Real-Time PCR technique was used to detect the relative expression of CcHSP70 gene in the striatum of Carrageenia crassifolia under high temperature stress at 28 鈩，

本文編號：1661496