Study On The Molecular Mechanism Of Rice Hull Color Genes OsBh4.2 And OsPhr1

As an important external organ of rice seeds,hulls not only affect the size and quality of the grains,but also play important biological functions such as resisting pests and predation by birds.The hulls of cultivated rice are mostly yellow,while the hulls of wild rice usually contain melanin and appear black.The black hull is often regarded as one of the typical characteristics of wild rice.However,the genetic mechanism of the domestication of black hull to yellow hull in rice is not clear.For this reason,our research group used Balilla and Mianhui 725（MH725）as parents in the early stage,and used genetic population construction and gene mapping analysis to screen out two gene loci that are the same as OsBh4 and OsPhr1 identified by the predecessors;Here,we deeply analyzed the gene structure,subcellular location,and protein interaction relationship of the site,and obtained the following results:1)The OsBh4 locus contains two open reading frames,named OsBh4.1 and OsBh4.2 respectively.OsBh4.1 has no difference between the parents Balilla and Mianhui725,but OsBh4.2 is significantly different.There is a 22bp deletion in Mianhui 725.In Balilla,another gene,OsPhr1,has a 18bp deletion in its coding region.The hulls of Balilla and Mianhui 725 rice were yellow,but the color of the hulls of F₁ hybrids changed to black after 14 days of flowering.2)The OsBh4.2 of Balilla and the OsPhr1 of Mianhui 725 were respectively amplified,and the corresponding overexpression genetic transformation vector was constructed using UBI as the promoter and transformed into Mianhui 725 and Balilla respectively.The results showed that the color of the rice hulls of the Balilla-positive plants transformed into OsPhr1 were all black,and the phenol reaction experiment further proved that the transformation was successful.Although OsBh4.2 was highly expressed in Mianhui 725-positive plants transformed with OsBh4.2,the color of rice hulls did not change and was still yellow.It is speculated that gene silencing may have occurred.Based on CRISPR/Cas9 technology,two knockout vectors of OsBh4.2 and OsPhr1 were constructed respectively,and Balilla and Mianhui 725 were genetically transformed correspondingly,and 6 OsBh4.2 mutants and 14 OsPhr1 mutants were obtained.3)Using qRT-PCR to detect the expression of black hull regulatory genes in rice hulls,the results showed that the expression of OsBh4.2 of Balilla and OsPhr1 of Mianhui725 increased at 14 days after anthesis,compared with 7 days after anthesis.Further analysis of the expression of flavonoid metabolism-related genes in rice hull 14 days after flowering showed that compared with the parents,most of the genes OsPAL,OsCHI,OsF3H,OsF3’H,and OsANS were up-regulated in the F₁ hybrid offspring.Black hull genes may positively regulate the metabolism of flavonoids.4)Transient expression analysis of rice protoplasts showed that both OsBH4.2 and OsPHR1 were located in chloroplasts.Bimolecular fluorescence complementation experiment（Bi FC）proved that OsBH4.2 and OsPHR1 interacted.Yeast two-hybrid experiment also proved that they interacted.Further through the yeast two-hybrid study of the interaction site,it was found that the four truncated mutants of protein OsBH4.2could not interact with OsPHR1,and the three truncated mutants of protein OsPHR1could all interact with OsBH4.2,therefore,the binding site needs to be further explored.This study determined that OsBh4.2 and OsPhr1 are both key genes involved in the regulation of rice hull color,and proved that there is an interaction between them.These results help to analyze the pigmentation patterns of rice hulls and provide direct molecular evidence for in-depth understanding of the domestication mechanism of rice hulls.