水稻轉錄因子OsbHLH061和OsbHLH068在非生物逆境下基因表現及功能性分析

The Genes Expression Profile and Functional Analysis of Rice Transcription Factor OsbHLH061 and OsbHLH068 under Abiotic Stresses

陳依甄(Yi-Jen Chen)

指導教授 ： 張孟基

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00456

摘要

非生物逆境，如乾旱、低溫、高溫、高鹽等逆境，都會影響水稻生長發育及產量損失。而轉錄因子所調控之基因表現影響水稻在非生物逆境下反應及耐受性相當重要。Apetala2/ethylene responsive factor (AP2/ERF) 及basic helix-loop-helix (bHLH) 分別為兩大轉錄因子基因家族。先前研究已知這兩群多基因家族在阿拉伯芥及水稻中，參與許多生長、生理及生物和非生物逆境等反應，但大部分的基因功能仍然未知。本研究首先利用生物資訊分析方法研究水稻AP2/ERF及bHLH轉錄因子基因在不同非生物逆境及組織專一性之基因表現圖譜，並搜尋出相對應之Tos17突變株，篩選出12個基因，再經由RT-PCR確定基因表現，最後篩選出OsERF106、OsbHLH035、OsbHLH061及OsbHLH068這4個基因。為了確定這些基因於逆境下的生理功能，進一步以功能性基因體學之研究方式，利用水稻Tos17突變株及與水稻OsbHLH061及OsbHLH068胺基酸序列相似之阿拉伯芥T-DNA突變株進行逆境耐性測試。目前已針對水稻及阿拉伯芥突變株以genotyping PCR及RT-PCR確定為同型結合突變且為knock down基因表現。由實驗結果顯示OsbHLH061基因之Tos17突變株發芽勢較低，在乾旱、高鹽及高溫逆境下比野生型較不具耐受性。另外OsbHLH061同源性之阿拉伯芥突變株atbhlh047在模擬高鹽及乾旱逆境下比野生型發芽率較低但呈現較長之根長，且將突變株成株以高鹽逆境處理發現存活率比野生型高。而異源表現(heterologous expression)水稻基因之阿拉伯芥轉殖株於高鹽逆境下根長比突變株短，初步推測OsbHLH061可能與AtbHLH047基因功能相似，未來仍需將OsbHLH061基因以互補方式(complementary rescue)轉入阿拉伯芥或水稻突變株中才能確定基因功能。OsbHLH068基因之Tos17突變株發芽勢也較低，在乾旱及高鹽逆境下比野生型較不具耐受性。另外OsbHLH068同源性之阿拉伯芥突變株atbhlh112在模擬高鹽及乾旱逆境下比野生型發芽率低但呈現較長之根長，突變株以高鹽逆境處理則存活率較高。以異源表現及互補水稻基因之阿拉伯芥轉殖株根長與突變株相同但比野生型長，此結果可能因OsbHLH068於轉殖株中基因表現較低所致。由以上研究顯示OsbHLH061及OsbHLH068基因確實參與非生物逆境反應，但於逆境下水稻及阿拉伯芥中表現結果不相似，此基因調控機制仍有待進一步釐清。本研究為後續提供目標基因進行水稻基因轉殖以研究基因功能及調控網絡奠下基礎，期望未來能以基因工程方式改良水稻對非生物逆境之耐受性及適應能力。

關鍵字

AP2/ERF ； bHLH ； 非生物逆境 ； 水稻Tos17突變株 ； 阿拉伯芥T-DNA突變株

並列摘要

Abiotic stresses such as drought, cold, heat, salt, have adverse effects on plant growth and crop yields. Transcription factors (TFs) are important for control the expression of target genes in response to abiotic stresses. The Apetala2/Ethylene Responsive Factor (AP2/ERF) and basic Helix-Loop-Helix (bHLH) are superfamily of TFs that regulate diverse processes of plant development and stress responses. However, the importance of specific AP2/ERF and bHLH TFs that are involved in the regulation of plant abiotic stress tolerance is still not known. In this study, we identified 4 putative stress-related TFs OsERF106, OsbHLH035, OsbHLH061 and OsbHLH068 by bioinformatics and gene expression patterns analysis. We used functional genomics approach to understand their roles under abiotic stresses, especially take advantage of rice Tos17 and corresponding Arabidopsis T-DNA mutants. We obtained the homozygous rice and Arabidopsis mutants for 4 TF genes by molecular characterization, including PCR-genotyping and RT-PCR analysis. The results showed that OsbHLH061 gene Tos17 rice mutant have low germination vigor. The Tos17 mutant of OsbHLH061 gene was much sensitive to drought, salt and heat stresses. In addition, AtbHLH047 T-DNA mutant line from Arabidopsis with corresponding homolog of OsbHLH061 gene, was sensitive to salt and mannitol stresses during seed germination, but opposite to seedling growth and aldult plants. Then, we heterologouly expressed OsbHLH061 gene in Arabidopsis. When compared to wild type plants, the transgenic plants exhibited short root length under salt stress. These results suggest that OsbHLH061 gene function may be similar to AtbHLH047 and it is worthy to apply complementary rescue approaches to further confirm OsbHLH061 genes function. OsbHLH068 gene Tos17 rice mutant showed low germination vigor. The Tos17 mutant of OsbHLH068 gene was sensitive to drought, salt stresses. In addition, AtbHLH112 T-DNA mutant line from Arabidopsis with corresponding homolog of OsbHLH068 gene, was sensitive to salt and mannitol stresses during seed germination, but opposite to seedling growth and aldult plants too. The heterologous expression and complementary rescue was carried out by overexpression OsbHLH061 gene in Arabidopsis wild type and mutant line. When compared to wild type plants and mutant line showed that the transgenic plants exhibited long root length under salt stress, because OsbHLH068 has low gene expression level. Taken together, the above study indicates that OsbHLH061 and OsbHLH068 TFs play significant roles in rice abiotic stress response. In the future, we will analyze the downstream stress-responsive genes under abiotic stress conditions to understand their genes regulatory network. In this study, we established a basis of these rice TFs candidate genes cloning and analyz their gene regulatory mechanism under abiotic stresses. It can be expected that genetic engineering TFs targeted gene transformation would provide a potential application in crop tolerance improvement.

並列關鍵字

AP2/ERF ； bHLH ； abiotic stress ； rice Tos17 mutant line ； Arabidopsis T-DNA mutant line

參考文獻

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