本文選題：皺波角叉菜　切入點(diǎn)：CaM　出處：《遼寧師范大學(xué)》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(yīng)答環(huán)境脅迫的信號通路及相應(yīng)蛋白質(zhì)功能的研究已成為熱點(diǎn)。作為機(jī)體受到一系列如高溫、干旱和嚴(yán)寒等非生物脅迫后大量表達(dá)的應(yīng)激蛋白,HSP70(Heat Shock protein 70)廣泛參與生物體內(nèi)變性蛋白質(zhì)的復(fù)性和降解,對機(jī)體的代謝調(diào)控和響應(yīng)環(huán)境脅迫具有重要的作用。隨著溫室效應(yīng)的加劇,災(zāi)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(fù)雜的調(diào)控機(jī)制來應(yīng)對熱刺激,鈣-鈣調(diào)素?zé)峒ば盘栟D(zhuǎn)導(dǎo)通路作為響應(yīng)熱脅迫主要的調(diào)控機(jī)制,可以調(diào)節(jié)HSP70的表達(dá)。本實(shí)驗(yàn)以經(jīng)濟(jì)海藻皺波角叉菜的葉狀體為實(shí)驗(yàn)材料,運(yùn)用RT-PCR技術(shù)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(jìn)行分析,結(jié)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(yù)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(jié)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(jié)合進(jìn)而來調(diào)節(jié)熱激信號轉(zhuǎn)導(dǎo)的下游事件,這為進(jìn)一步研究皺波角叉菜的熱激信號轉(zhuǎn)導(dǎo)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(yīng)特點(diǎn),本研究采用Real-Time PCR技術(shù)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(dá)量的變化。并將CcHSP70基因轉(zhuǎn)入大腸桿菌內(nèi)進(jìn)行原核表達(dá)與分離純化,隨后檢測了CcHSP70蛋白質(zhì)的ATPase活性,測定45℃高溫與10℃冷脅迫下轉(zhuǎn)pET28a-CcHSP70大腸桿菌的生長情況。結(jié)果顯示:CcHSP70基因的表達(dá)受到高溫脅迫與冷脅迫的顯著誘導(dǎo),在持續(xù)28℃高溫脅迫下,CcHSP70的表達(dá)量在4 h時達(dá)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(dá)量與對照組相比達(dá)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(jié)合和釋放。在45℃高溫與10℃冷脅迫下CcHSP70的過表達(dá)提高了大腸桿菌的抗高溫能力與耐冷性。結(jié)果表明CcHSP70受熱激與冷脅迫的誘導(dǎo)表達(dá),在高溫脅迫與冷脅迫中維持了細(xì)胞蛋白的穩(wěn)定,對細(xì)胞具有重要的保護(hù)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