利用CRISPR／Cas9基因編輯技術進行水稻OsCDPK1的功能失活突變

Loss-of-function mutation of OsCDPK1 in rice by CRISPR/Cas9 gene editing

陳彥伯(Yan-Bo Chen) ； 董柏宏(Bo-Hong Dong) ； 侯新龍(Shin-Lon Ho)

《嘉大農林學報》 16卷2期 (2019/12) Pp. 53-66

摘要

鈣調蛋白質激酶（CDPKs, calcium-dependent protein kinases）是植物細胞內Ca^(2+)濃度變化的感測器（sensor）之一，其蛋白質激酶的活性被Ca^(2+)所活化，進而磷酸化目標蛋白質的絲胺酸（Serine）及息寧胺酸（Threonine）胺基酸，因此是屬於Ser/Thr蛋白質激酶。OsCDPK1過量表現（OsCDPK1-overexpressing）對幼苗生長及種子大小具有負向的影響；且OsCDPK1-RNAi（RNA interference）基因靜默（gene silencing）的轉殖株（Ri-1）幼苗成長較快且穀粒較大，葉片顏色為淡綠色，植株型態較細長。為了獲得穀粒較大但不帶轉基因的OsCDPK1突變株，本研究利用叢集有規律間隔的短迴文重複序列（clustered regularly interspaced short palindromic repeats, CRISPR）/ CRISPR- 結合蛋白9（CRISPR-associated protein 9, Cas9）基因體編輯的方法，進行OsCDPK1的基因功能失活突變，將重組完成的sgRNA-OsCDPK1/Ubi::Cas9 binary vector以農桿菌為媒介轉殖到水稻TNG67癒傷組織中，共獲得109個轉殖成功的瘉傷組織系，其中有24個系成功誘導成植株，誘導成功率為22%。純化轉殖系的基因組（genomic）DNA後，利用PCR將目標序列增殖並選殖到T-vector，目前定序的T_0植株系中，發現有三個植株系在OsCDPK1基因的轉譯起始點下游第79個核苷酸G密碼子缺失；一個植株系於轉譯起始下游第80個核甘酸插入一個A密碼子；兩個植株系於轉譯起始下游第81個核甘酸插入一個T密碼子，皆造成密碼讀框被改變，轉譯提早終止。推測成功獲得了3個不同之基因編輯OsCDPK1功能失活的突變株。

關鍵字

水稻 ； OsCDPK1 ； CRISPR／Cas9 ； 基因編輯

並列摘要

CDPKs (calcium-dependent protein kinases) is one of the plant Ca^(2+) sensors, their kinase activities are activated by Ca^(2+) that subsequently phosphorylation on serine or threonine residues in the targeted proteins. Therefore, they are belong to as a Ser/Thr protein kinases. Our studies found that the seedling growth and seed size are negatively and positively affected in transgenic OsCDPK1-overexpressing and OsCDPK1-RNAi rice plants, respectively. To generate a rice plant of which could produces a larger seed than that of the wild-type but did not carry the transgene in the genome, the strategy of Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) gene editing was conducted in this study for loss of function mutation of OsCDPK1. The sgRNA-OsCDPK1/Ubi::Cas9 binary vector was conducted and transformed by Agrobacterium-mediated gene transformation of rice calli. Twenty-four out of 109 transformed calli were successfully regenerated into transgenic plants and the regeneration rate was 22%. The genomic DNAs were purified from those different transgenic lines followed by amplified of the targeted gene OsCDPK1 coding region by PCR and cloned into T-vector. After DNA sequencing, three individual transgenic lines were found that from the downstream of the OsCDPK1 translational start codon, the 79^(th) nucleotide of G was deleted. We also found that there were one transgenic line was showed single nucleotide insertion of A in the position of 80^(th) nucleotide, and two lines revealed that a T nucleotide insertion in 81^(th) base. All of these mutants were as frame-shift mutation, which would result in the premature termination codon during translation. Altogether, by the CRISPR/Cas9 gene editing, there were three transgenic lines that were successfully edited in OsCDPK1 coding region and resulted in loss-of-function mutation.

並列關鍵字

rice ； OsCDPK1 ； CRISPR/Cas9 ； gene editing