鈣離子響應(yīng)蛋白GCaMP6s在稻瘟菌中的異源表達

林艾佳 吳奇 倪瑞蓮 陳恒 衛(wèi)冕 李成云 王一

摘要 稻瘟病嚴(yán)重威脅全球水稻的產(chǎn)量，鈣離子信號通路參與了稻瘟菌生長、發(fā)育和致病，了解這些過程中菌株細(xì)胞質(zhì)中鈣離子濃度的變化對稻瘟病的防治具有重要意義。GCaMP6s作為一種細(xì)胞質(zhì)鈣離子感受蛋白在動物和植物中已有大量應(yīng)用。本研究利用不同稻瘟菌菌株構(gòu)建了GCaMP6s基因的異源表達菌株，在0.01 g/mL鈣離子處理下，GCaMP6s在稻瘟菌菌絲和分生孢子中穩(wěn)定表達。此外，GCaMP6s異源表達菌株與野生型菌株的菌落直徑和產(chǎn)孢量沒有差異，表明GCaMP6s基因的異源表達不影響稻瘟菌的菌絲生長和產(chǎn)孢。本研究獲得了GCaMP6s異源表達的稻瘟菌菌株，為后續(xù)研究稻瘟菌細(xì)胞質(zhì)中鈣離子濃度變化與菌株生長發(fā)育和脅迫響應(yīng)提供研究材料。

關(guān)鍵詞 稻瘟菌; 異源表達; GCaMP6s

中圖分類號： S435.111.41

文獻標(biāo)識碼： A

DOI： 10.16688/j.zwbh.2021023

Abstract Rice blast caused by Magnaporthe oryzae is a serious threat to the global rice production. Calcium signaling pathway is involved in the growth， development and pathogenicity of M.oryzae. Understanding the changes of Ca2+ concentration in the cytoplasm of M.oryzae at three different stages is of great significance for control of rice blast. As a cytoplasmic Ca2+ sensor protein， GCaMP6s has been widely used in animals and plants. In this study， heterologous expression strains containing GCaMP6s were constructed using M.oryzae YN153 and YN157 strains. Under 0.01 g/mL Ca2+ treatment， GCaMP6s was stably expressed in the mycelia and conidia of M.oryzae. In addition， by comparing the colony diameter and sporulation between the heterologous expression strains of GCaMP6s and the wild type strains， we found that there was no difference between the two strains， indicating that the heterologous expression of GCaMP6s gene did not affect the mycelial growth and sporulation of M.oryzae. In this study， we obtained the heterologous expression of GCaMP6s in M.oryzae， which can provide research materials for further study on the changes of calcium ion concentration in the cytoplasm， and the growth， development and stress response of M.oryzae.

Key words Magnaporthe oryzae; heterologous expression; GCaMP6s

細(xì)胞內(nèi)游離鈣是重要的第二信使，在生物體生長發(fā)育、轉(zhuǎn)錄調(diào)控、細(xì)胞代謝等方面具有重要作用[1]。近年來有關(guān)鈣離子的報道在動植物和微生物免疫研究方面較多。在真核微生物中，鈣離子鈣調(diào)蛋白鈣調(diào)磷酸酶（calcium-calmodulin-calcineurin）級聯(lián)通路對病原真菌生長發(fā)育，特別是其毒力和侵染能力具有重要影響[2]，與病原真菌鈣離子穩(wěn)態(tài)、脅迫響應(yīng)、細(xì)胞壁完整性等關(guān)系密切。例如，在鏈格孢Alternaria alternata中，鈣調(diào)磷酸酶基因Cna1影響菌絲形態(tài)和分生孢子成熟[3];在大麗輪枝菌Verticillium dahliae中鈣調(diào)磷酸酶基因Crz1與菌核形成相關(guān)[4]。此外，在玉米黑粉菌Ustilago maydis和核盤菌Sclerotinia sclerotiorum中細(xì)胞周期蛋白依賴性激酶1（cyclin-dependent kinase 1， Cdk1）通過調(diào)控鈣離子的濃度控制菌株有絲分裂，最終影響了菌株的有性生殖能力[56]。……