| Rice is one of the major food crops in the world.With the continuous growth of the global population and the decrease of the cultivated land area,the improvement of rice yield is of great significance for ensuring world food security.Practice has proven that the combination of ideal plant type and heterosis utilization is an important way to further improve rice yield.Tiller angle is an important part of the ideal plant type of rice,which directly affects the canopy structure of rice,thus affects the photosynthetic efficiency of leaves,and finally affects the yield and quality of rice.Therefore,it is necessary and crucial to dig out genes regulating tiller angle and study their functions,which will be of great significance to the cultivation of ideal plant type rice.In this study,we cloned the rice SBP transcription factor E204 through the reverse genetics approach.The gene overexpression vector and CRISPR/Cas9 gene editing vector were constructed and the germplasm of Zhonghua11(ZH11)embryogenic callus was used as the genetic transformation receptor to obtain positive transgenic rice.The agronomic traits of the transgenic lines were investigated in combination with RNA expression analysis.Finally,the structure and function of the E204-interacting proteins screened out through yeast twohybrid system were analyzed,to determine the function and regulatory mechanism of E204.The main results of this study were as follows:(1)Phenotypic observation: The tiller angle of E204 overexpressed transgenic lines(OTL)was large and the plant type was scattered.However,the tiller angle of ZH11 was small and the plant type was erect,indicating that the overexpression of E204 would lead to the increase of tiller angle in rice.Through observing the longitudinal section of the tiller base,the growth of the far-land side and near-land side of the tiller base of ZH11 was unbalanced,while the growth of the two sides of the OTL tiller base was balanced,indicating that the overexpression of E204 could break the growth imbalance of the two sides of the tiller base and increase the tiller angle.(2)Real-time PCR analysis: The expression of E204 in OTL was higher than that of ZH11 in different rice tissues.In ZH11,the expression of E204 in the near-land side of the tiller base was higher than that of the far-land side.However,in the tiller base of OTL,the gene expression in the far-land side was higher than the near-land side.In addition,Os PIN2,which had been reported to regulate tiller angle in rice,showed higher expression level at the tiller base of OTL than in ZH11,indicating that Os PIN2 might be downstream to E204.(3)Investigation of the agronomic traits of CRISPR/Cas9 transgenic lines(CTL)showed that tiller angle,number of stems and tillers and grain length of CTL were the same as ZH11.However,the expression of E204 in tiller base of CTL was lower than that of ZH11,suggesting that the CTL material constructed in this study was correct.Therefore,it was speculated that an unknown redundant tiller angle regulating gene in ZH11 might still be functional.Therefore,the tiller angle of rice remained unchanged when E204 was mutated and lost its function.(4)Yeast two-hybrid screening:66 E204-interacting-proteins were screened out and 7proteins with the highest frequency in the positive clones were selected for analysis.The results showed that the interacting proteins IP1,IP2 and IP7 regulated the expression of hormone response genes in Arabidopsis and controlled the cell proliferation and elongation of the meristem and root tip.Therefore,it could be speculated that in rice,E204 indirectly regulated the expression of auxin transporter gene Os PIN2 by interacting with these three proteins,thus influenced the cell proliferation and elongation of stem meristem,and finally regulated the increase of tiller angle in rice.The knowledge gained in this study of functional analysis of E204,a tiller angle regulating gene,could provide a theoretical basis for the cultivation of rice varieties with ideal plant type in the future. |
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