發(fā)布時(shí)間：2018-03-17 08:25

  本文選題：葉用萵苣　切入點(diǎn)：Hsp　出處：《中國農(nóng)業(yè)科學(xué)》2017年08期 　論文類型：期刊論文

【摘要】：【目的】通過克隆Hsp70相關(guān)基因,并利用VIGS分析葉用萵苣熱脅迫下Hsp70表達(dá)量和形態(tài)變化,為解析熱激蛋白基因Hsp70在高溫脅迫下的響應(yīng)機(jī)制及分子機(jī)理奠定基礎(chǔ)�！痉椒ā客ㄟ^同源克隆及RACE技術(shù),獲得葉用萵苣熱激蛋白LsHsp70-2711的cDNA全長序列,利用實(shí)時(shí)熒光定量PCR(qRT-PCR)分析該基因在不同溫度和不同高溫時(shí)間下的葉用萵苣熱敏品種‘P-S11’和耐熱品種‘G-S59’的表達(dá)差異,確定基因和高溫的相關(guān)性。根據(jù)VIGS技術(shù),構(gòu)建p TRV-LsHsp702711瞬時(shí)沉默載體,轉(zhuǎn)化農(nóng)桿菌GV1301,注射法侵染葉用萵苣葉片,三周后經(jīng)PCR鑒定得到陽性植株。對照組和陽性組在基因表達(dá)和形態(tài)上進(jìn)行對比,對照植株和陽性植株熱脅迫和干旱處理后再次分析LsHsp70-2711的表達(dá)特性,觀測形態(tài)變化�！窘Y(jié)果】LsHsp70-2711 cDNA全長為2 226 bp,開放閱讀框?yàn)? 154 bp,編碼718個(gè)氨基酸,與擬南芥(NP_187864.1)、番茄(NP_001266213.1)、水母雪蓮花(AAB99745.1)等物種的Hsp70同源性達(dá)到80%以上,證明此基因?qū)儆贖sp70家族。根據(jù)qRT-PCR結(jié)果,高溫脅迫下該基因在兩個(gè)品種中的表達(dá)均上調(diào),在耐熱品種中總體表達(dá)水平均高于熱敏品種,耐熱品種LsHsp70-2711的表達(dá)量最大值出現(xiàn)在42℃、60 min,37℃、60 min時(shí)熱敏品種基因表達(dá)量達(dá)最大值,且在42℃高溫下熱敏品種‘P-S11’中基因表達(dá)隨著脅迫時(shí)間的增加受到抑制,而耐熱品種‘G-S59’則能長時(shí)間保持較高的表達(dá)水平,此結(jié)果和田間兩品種間耐熱差異表現(xiàn)相符合。亞細(xì)胞定位顯示,LsHsp70-2711主要在細(xì)胞質(zhì)中。將構(gòu)建好的載體侵入葉用萵苣,鑒定后獲得陽性植株。由定量PCR結(jié)果可知,未進(jìn)行脅迫處理的陽性植株與對照株相比,LsHsp70-2711表達(dá)量下降,莖長明顯增長。熱脅迫和干旱處理后的陽性植株LsHsp70-2711表達(dá)量顯著低于對照植株,高溫處理對LsHsp70-2711的影響大于干旱脅迫�！窘Y(jié)論】LsHsp70-2711屬于Hsp70基因家族,其與葉用萵苣耐熱性相關(guān)。研究結(jié)果為解析葉用萵苣熱激蛋白LsHsp70-2711在葉用萵苣高溫抽薹方面的功能提供了理論支持。
[Abstract]:[objective] to clone Hsp70 related genes and analyze the changes of Hsp70 expression and morphology under heat stress in lettuce by VIGS. In order to elucidate the response mechanism and molecular mechanism of heat shock protein gene Hsp70 under high temperature stress. [methods] the full-length cDNA sequence of heat shock protein LsHsp70-2711 in lettuce was obtained by homologous cloning and RACE technique. Using real-time fluorescence quantitative PCR qRT-PCR to analyze the difference of expression of the gene in heat sensitive variety of lettuce under different temperature and time, P-S11 'and heat-resistant variety GS59', to determine the correlation between gene and high temperature. The transient silencing vector of p TRV-LsHsp702711 was constructed and transformed into Agrobacterium tumefaciens GV1301. The lettuce leaves were infected by injection method. The positive plants were identified by PCR three weeks later. The gene expression and morphology of the control group and the positive group were compared. After heat stress and drought treatment in control and positive plants, the expression of LsHsp70-2711 was analyzed again, and morphological changes were observed. [results] the total length of LsHsp70-2711 cDNA was 2 226 BP, the open reading frame was 2 154 BP, encoding 718 amino acids. The homology of Hsp70 was more than 80% with other species such as Arabidopsis thaliana 187864.1, Tomato NP001266213.1 and AAB99745.1), which proved that this gene belonged to the Hsp70 family. According to the results of qRT-PCR, the expression of this gene in both cultivars was up-regulated under high temperature stress. The total expression level of heat resistant varieties was higher than that of heat sensitive varieties, and the maximum expression of LsHsp70-2711 in heat resistant varieties reached the maximum at 42 鈩，

本文編號：1623933