發(fā)布時間：2018-11-16 15:54

【摘要】：以‘徐稻4號’為材料,構(gòu)建OsCaMs的瞬時表達載體,利用生物信息學(xué)和RT-PCR方法設(shè)計OsCaMs定量引物5個,鑒定參與ABA誘導(dǎo)的OsCaMs基因并分析其生理功能,為進一步揭示ABA信號轉(zhuǎn)導(dǎo)核心組分的研究奠定基礎(chǔ)。結(jié)果顯示:(1)水稻OsCaMs的5個家族基因的CDS等長,均為450bp,ABA(100μmol·L~(-1))處理能夠明顯誘導(dǎo)OsCaM1-1和OsCaM1-2的表達。(2)利用瞬時表達載體將表達熒光蛋白的OsCaM1-1-YFP和OsCaM1-2-YFP通過PEG方法轉(zhuǎn)化到原生質(zhì)體中,激光共聚焦分析顯示,這2個基因均定位于細胞核、細胞質(zhì)和細胞膜中;并構(gòu)建了OsCaM1-1和OsCaM1-2的干擾載體dsOsCaM1-1和dsOsCaM1-2。(3)經(jīng)ABA誘導(dǎo)的水稻原生質(zhì)體抗氧化保護酶APX和SOD活性分別顯著提高35%和31%;原生質(zhì)體瞬時表達和瞬時沉默體系分析表明,OsCaM1-1和OsCaM1-2均能夠影響抗氧化保護酶APX和SOD的活性,其中原生質(zhì)體中瞬時過表達這2個基因?qū)PX和SOD活性上調(diào)達到1.15~1.45倍,瞬時干擾這2個基因?qū)PX和SOD活性下調(diào)達到25%~30%。(4)外源H_2O_2(10mmol·L~(-1))預(yù)處理水稻幼苗可誘導(dǎo)OsCaM1-1和OsCaM1-2基因表達量上調(diào),并且OsCaM1-2能夠誘導(dǎo)水稻原生質(zhì)體中H_2O_2的積累。(5)原生質(zhì)體瞬時表達分析發(fā)現(xiàn),在水稻原生質(zhì)體中瞬時表達OsCaM1-1和OsCaM1-2可分別誘導(dǎo)OsrbohB和OsrbohE的表達;而且在水稻原生質(zhì)體中瞬時表達OsrbohB和OsrbohE可分別誘導(dǎo)OsCaM1-1和OsCaM1-2的表達,表明OsCaMs與Osrbohs之間存在正反饋調(diào)節(jié)機制。研究表明,OsCaM1-1和OsCaM1-2為水稻參與ABA信號轉(zhuǎn)導(dǎo)中具有調(diào)控抗氧化保護作用的同源基因;OsCaM1-1和OsCaM1-2不僅受ABA誘導(dǎo),也受H_2O_2誘導(dǎo),而且ABA是通過H_2O_2進行調(diào)控。
[Abstract]:The transient expression vector of OsCaMs was constructed by using Xudao 4 as the material. Five OsCaMs quantitative primers were designed by bioinformatics and RT-PCR methods. The OsCaMs gene involved in ABA induction was identified and its physiological function was analyzed. To lay a foundation for the further study of the core components of ABA signal transduction. The results showed that: (1) the CDS of the five family genes of rice OsCaMs was 450bp. ABA (100 渭 mol L ~ (-1) could significantly induce the expression of OsCaM1-1 and OsCaM1-2. (2) OsCaM1-1-YFP and OsCaM1-2-YFP expressing fluorescent protein were transformed into protoplasts by PEG. Laser confocal analysis showed that the two genes were located in the nucleus, cytoplasm and cell membrane. The interference vectors of OsCaM1-1 and OsCaM1-2, dsOsCaM1-1 and dsOsCaM1-2. (3), were constructed. The antioxidant protective enzymes APX and SOD induced by ABA in rice protoplasts were significantly increased by 35% and 31%, respectively. The transient expression of protoplasts and transient silencing system showed that both OsCaM1-1 and OsCaM1-2 could affect the activities of antioxidant protective enzymes APX and SOD. The transient overexpression of these two genes in protoplasts increased the activity of APX and SOD by 1.15 ~ 1.45 times. (4) exogenous H_2O_2 (10mmol L-1) pretreatment could induce the up-regulation of OsCaM1-1 and OsCaM1-2 gene expression in rice seedlings. OsCaM1-2 could induce the accumulation of H_2O_2 in the protoplasts of rice. (5) the transient expression of OsCaM1-1 and OsCaM1-2 in the protoplasts of rice could induce the expression of OsrbohB and OsrbohE, respectively. Moreover, transient expression of OsrbohB and OsrbohE in rice protoplasts could induce the expression of OsCaM1-1 and OsCaM1-2, respectively, indicating that there was a positive feedback regulation mechanism between OsCaMs and Osrbohs. The results showed that OsCaM1-1 and OsCaM1-2 were the homologous genes involved in ABA signal transduction. OsCaM1-1 and OsCaM1-2 were induced not only by ABA but also by H_2O_2, and ABA was regulated by H_2O_2.
【作者單位】： 南京農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院;
【基金】：基金項目:中央高�；緲I(yè)務(wù)費(KYZ201157)
【分類號】：Q943.2
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本文編號：2335943