【摘要】：以‘云香’水仙為材料,采用PCR技術(shù)分離中國水仙WRKY轉(zhuǎn)錄因子家族成員NtWRKYY2(GenBank登錄號為KX056496),其開放閱讀框(ORF)長度為867bp,編碼289個氨基酸。氨基酸序列比對及系統(tǒng)進(jìn)化樹分析顯示,NtWRKYY2編碼蛋白含1個WRKY結(jié)構(gòu)域和C2H2鋅指結(jié)構(gòu)(Csx4Cx23HxH),屬于第Ⅱd類WRKY轉(zhuǎn)錄因子。實時熒光定量PCR(qRT-PCR)分析顯示,NtWRKYY2在‘云香’水仙應(yīng)對鹽脅迫中顯著上調(diào)表達(dá)。利用InFusion克隆技術(shù)成功構(gòu)建過表達(dá)載體pMDC140-NtWRKYY2,并采用農(nóng)桿菌介導(dǎo)葉盤法轉(zhuǎn)化煙草,獲得11株卡那霉素抗性植株,轉(zhuǎn)化子進(jìn)一步PCR檢測結(jié)果顯示,其中有8株目的基因已成功導(dǎo)入煙草基因組中,轉(zhuǎn)化率為72%。鹽脅迫處理和葉綠素?zé)晒鈪?shù)分析顯示,鹽脅迫處理后NtWRKYY2過表達(dá)的轉(zhuǎn)基因煙草萎蔫和黃化程度小于野生型植株,Fv/Fm值下降幅度小于野生型植株。研究表明,NtWRKYY2過表達(dá)的轉(zhuǎn)基因煙草具有抵抗鹽脅迫的能力。該研究為水仙抗鹽轉(zhuǎn)基因育種提供備選目的基因。
[Abstract]:NtWRKYY2 (GenBank accession number: KX056496), a member of the WRKY transcription factor family of Narcissus sinensis, was isolated by PCR from Narcissus sinensis. The open reading frame (ORF) length was 867 BP, encoding 289 amino acids. Amino acid sequence alignment and phylogenetic tree analysis showed that NtWRKYY2 encoded protein contained a WRKY domain and C2H2 zinc finger structure (Csx4Cx23HxH), which belonged to the second day WRKY transcription factor. Real time fluorescence quantitative PCR (qRT-PCR) analysis showed that NtWRKYY2 significantly up-regulated the expression of Narcissus in response to salt stress. The overexpression vector pMDC140-NtWRKYY2, was successfully constructed by InFusion cloning technique and transformed into tobacco by Agrobacterium tumefaciens leaf disc method. Eleven kanamycin resistant plants were obtained. Further PCR detection of the transformants showed that the transgenic plants were resistant to kanamycin. Eight of them were successfully introduced into the tobacco genome, and the transformation rate was 72%. The results of salt stress and chlorophyll fluorescence analysis showed that the degree of wilting and yellowing of transgenic tobacco with NtWRKYY2 overexpression after salt stress was less than that of wild type plants. The results showed that transgenic tobacco with NtWRKYY2 overexpression had the ability to resist salt stress. The study provides alternative target genes for salt-tolerant transgenic breeding of narcissus.
【作者單位】： 福建農(nóng)林大學(xué)園藝學(xué)院;福建農(nóng)林大學(xué)園藝植物遺傳育種研究所;
【基金】：福建省種業(yè)創(chuàng)新與產(chǎn)業(yè)化工程項目(K8114001B)
【分類號】：Q943.2;S682.21

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