藥用野生稻抗逆相關(guān)基因的克隆及功能研究.pdf

1、I 摘 要 藥用野生稻（Oryza officinalis）攜帶多種生物和非生物脅迫的抗（耐）性基因，是水稻遺傳改良的重要基因庫(kù)。sHSP、ADF 和 GST 基因在生物體防衛(wèi)反應(yīng)和逆境脅迫應(yīng)答中起著重要的作用。本研究從藥用野生稻（CC 染色體組）干旱處理轉(zhuǎn)錄組測(cè)序結(jié)果中篩選出 3 個(gè)表達(dá)差異顯著的 sHSP、ADF 和 GST 家族預(yù)測(cè)基因，分別命名為OoHsp20-3、OoADF1 和 OoGST1，對(duì)它們進(jìn)行基因功能探究。

2、利用同源克隆法擴(kuò)增出目的基因完整 CDS， 測(cè)序結(jié)果表明， OoHsp20-3、 OoADF1和 OoGST1 完整的 ORF 長(zhǎng)度分別為 522bp、453bp 和 690bp，分別編碼 173、150 和229 個(gè)氨基酸殘基， 在藥用野生稻基因組中， OoHsp20-3、 OoADF1 和 OoGST1 編碼區(qū)長(zhǎng)度分別為 526bp、459bp、700bp，均沒有內(nèi)含子。 利用 qRT-PCR 技術(shù)，研究了 OoHsp20-3、Oo

3、ADF1 和 OoGST1。結(jié)果顯示，OoHsp20-3、 OoADF1 和 OoGST1 在所檢測(cè)的 7 個(gè)組織中均表達(dá)。 OoHsp20-3 和 OoADF1均在四葉期幼苗的根中表達(dá)量最高，而在抽穗期的葉鞘中表達(dá)量最高。OoGST1 在四葉期幼苗的葉鞘中表達(dá)量最高，抽穗期時(shí)則在葉片中表達(dá)量最高。此外還研究了OoHsp20-3、OoADF1 和 OoGST1 分別在干旱、高溫、低溫和高鹽濃度脅迫下的表達(dá)量變化特點(diǎn)。結(jié)果表明，OoHsp

4、20-3、OoADF1 和 OoGST1 在高溫、干旱和高鹽濃度脅迫時(shí)表達(dá)量上調(diào)。OoHsp20-3 在高溫和高鹽濃度脅迫時(shí)表達(dá)量較未處理的對(duì)照分別上調(diào)了 240 倍和 1000 倍，OoADF1 在干旱和高鹽濃度脅迫時(shí)表達(dá)量較對(duì)照分別上調(diào)了 17.9 倍和 40 倍，OoGST1 在高溫和高鹽濃度脅迫時(shí)表達(dá)量較對(duì)照分別上調(diào)了 3.6倍和 6 倍。 將 OoHsp20-3、 OoADF1 和 OoGST1 完整的 CDS 融合到原核表達(dá)

5、載體 pET-32a(+)或 pET-28a(+)，通過 IPTG 誘導(dǎo)及 SDS-PAGE 檢測(cè)，結(jié)果表明這三個(gè)基因編碼的蛋白分子量分別約為 14kD、16kD 和 23kD。OoHsp20-3 在 IPTG 終濃度分別為 1.0mM （pET-32a(+)）和 0.5 mM（pET-28a(+)）時(shí)誘導(dǎo)蛋白表大量最大；OoADF1 和 OoGST1在 IPTG 終濃度分別為 0.2mM 和 0.8mM 時(shí)誘導(dǎo)蛋白表大量最大。細(xì)胞破碎

6、顯示OoHsp20-3、OoADF1 和 OoGST1 編碼的蛋白主要以包涵體的形式存在。對(duì)轉(zhuǎn)入重組子及空載體的大腸桿菌 E.coli BL21(DE3)菌株分別進(jìn)行 500 mM NaCl 和 600 mM NaCl液體和固體培養(yǎng)脅迫處理， 與轉(zhuǎn)入空載體的菌株相比， 均能夠更好的耐受高濃度 NaCl的脅迫，表明 OoHsp20-3、OoADF1 和 OoGST1 對(duì)鹽脅迫有一定的響應(yīng)能力，能夠保III Cloning and Func

7、tional Analysis of Genes Resistant to Adversity in Oryza officinalis Li Hongmei (Agricultural College, South China Agricultural University, Guangzhou, 510642, China) Abstract: Oryza officinalis contains a variety of gene

8、s resistant to biotic and abiotic stresses, and is an important genetic pool for rice genetic improvement. sHSP, ADF and GST genes play important roles in defense response and adversity stress response. From transcriptom

9、e data of Oryza officinalis seedlings under 20% PEG drought stress, three genes, OoHsp20-3, OoADF1 and OoGST1 were detected to be significantly regulated and selected for further gene functional studies. Using the homolo

10、gy-based cloning method, the complete CDS of OoHsp20-3, OoADF1 and OoGST were amplified and sequenced, respectively. The results showed that the coding regions of OoHsp20-3, OoADF1 and OoGST1 are 526 bp and 459 bp and 67

11、8 bp-length, respectively. The complete open reading frame (ORF) of OoHsp20-3, OoADF1 and OoGST1 are 522, 453 and 567 bp-length, respectively, encoding 174, 151, and 189 amino acid residues, respectively. OoHsp20-3, OoAD

12、F1 and OoGST1 are intronless. The specific patern of OoHsp20-3, OoADF1 and OoGST1 in different tissues of four leaves stage seedlings and heading stage of Oryza officinalis were studied by real-time fluorescence quantita

13、tive PCR technology. The results showed that the three genes were all expressed in all the seven tissues. OoHsp20-3 and OoADF1 had highest expression in root during four leaves stage and in the leaf sheath during heading

14、 stage, respectively. OoGST1 had highest expression in leaf sheath during four leaves stage and in leaf during heading stage. The expression changes of OoHsp20-3, OoADF1 and OoGST1 under drought, high temperature, low te

15、mperature and high salt stresses were also studied in four leaves stage seedlings. The fold change of OoHsp20-3 expression were 240 times and 1000 times under drought and high salt stresses, respectively. The fold change