Function Analysis Of Hd2 UORF In Rice

Heading date of rice（Oryza sativa）,also known as flowering time,refers to the time from sowing to heading,which directly determines the utilization rate of light and the yield of rice.As a quantitative trait,the heading date of rice is regulated by exogenous factors and endogenous genes,many of which are regulated by photoperiod.They form a complex regulatory network.China has a vast territory,and there is a big difference in photoperiod time between the north and the south,so it is very necessary to breed rice varieties suitable for different light conditions,which is of great significance to expand the planting area of rice in China and improve the yield of rice.Hd2/PRR37/Ghd7.1（Heading date 2/Pseudoresponse Regulator 37/Grain number,Plant height,and Heading date 7.1）is the main gene that controls the heading date of rice in Northeast China.It reduces the expression of florigen genes Hd3a（Heading date 3a）and RFT1（Rice Flowering Lcucs T1）by inhibiting the expression of flowering-promoting factor Ehd1（Early heading date 1）,thereby inhibiting flowering in rice.The upstream ORF（u ORF,Upstream Open Reading Frame）exists in the 5’UTR（5’Untranslated Region）region of the gene,and has the effect of inhibiting the translation of the main downstream ORFs（p ORFs,Primary Open Reading Frames）.In this study,by analyzing the plant u ORF database（http://uorflight.whu.edu.cn/）,it was determined that there were three u ORFs in the 5’UTR of the rice Hd2 gene.By editing u ORF using CRISPR/Cas9,the acquired mutant plants with delayed heading date confirmed that Hd2 u ORF regulated flowering time in rice.The main research results are as follows:（1）Using CRISPR/Cas9,we designed targets for three u ORFs of Hd2,constructed gene editing plasmids,transformed rice variety Songjing 2（SJ2）,and obtained 12 antibiotics-resistant transgenic plants,including 4 homozygous mutants,4 heterozygous mutants and 4 unedited plants.（2）To analyze the function of Hd2 u ORF,the heading date of SJ2 and T₂homozygous mutants（hd2 uorf）was investigated.The mutants exhibited delayed flowering of rice by 4.6-11.2 days.The investigation of other agronomic traits（tiller number,plant height,panicle length,primary branch number,secondary branch number,grain number,seed setting rate and yield per plant）showed that compared with SJ2,hd2 uorf had increased panicle length,primary branch number,grain number and yield per plant.（3）The expression differences of Hd2,Ehd1 and florigen genes Hd3a and RFT1 in SJ2 and hd2uorf were detected by q RT-PCR.The results showed that the functional loss of u ORFs in hd2 uorf did not affect Hd2 transcription,but the expression levels of Ehd1,Hd3a and RFT1 were significantly lower than those in SJ2,consistent with the delayed flowering phenotype of hd2 uorf.（4）To determine whether the loss of function of Hd2 u ORFs affects the translation of p ORF of a gene,the protoplast transient transformation system was used to compare the effect of Hd2 u ORF and hd2 uorf on the translation level of downstream Firefly Luciferase（LUC）.The results showed that Hd2 u ORF significantly inhibited the expression of LUC,and the replacement of hd2 uorf could alleviate the inhibition of LUC expression to varying degrees,confirming that Hd2 u ORF functions by inhibiting gene translation.In summary,this study found that there are three u ORFs in the 5’UTR region of Hd2,and confirmed that they play a role in regulating heading date by inhibiting the translation level of Hd2p ORF.In addition,this study developed a method to delay flowering in rice by editing the u ORF region of the rice flowering inhibitor,which saves time and effort compared with traditional breeding.Using this method,the u ORFs of other flowering-promoting factors and flowering-inhibiting factors can be edited to achieve the purpose of regulating the heading date of different rice varieties.