Molecular Identification And Preliminary Analysis Of Brassinosteroid-related Mutants And Transgenic Rice

Brassinosteroids(BR),known as the sixth major phytohormone,are the polyhydroxylated steroidal hormones that are ubiquitously present in the plant kingdom.BRs are involved in a series of physiological and biochemical events and play essential roles in plant growth and development.At present,a near complete BR signal transduction pathway from the cell surface receptor BRI1 to the nuclear transcription factor BZR1 and BES1 has been established in the dicotyledonous model plant Arabidopsis.However,many important components of BR signal transduction pathway have yet to be cloned and functionally characterized in rice,which is one of the most important staple crop and monocotyledonous model plant.In this study,six key genes involving in BR pathway were selected,including BR biosynthesis gene DWF4,BR receptor encoding gene D61 and four genes of the BZR transcription factor family.We analyzed the effect of BR biosynthesis and signaling on rice architecture,grain appearance,eating and cooking quality by using BR insensitive mutants and generated transgenic rice,including both overexpression lines and knock-out lines.This study has laid the foundation for dissecting BR functions,culturing new rice varieties with high yield and elite quality via genetic manipulation of BR pathway.The major results of this study are as following.1.Rice mutants of key genes in BR signaling pathway,including hzrl,bzr4-1,bzr4-2,d61-1 and d61-2,were collected for a systematic analysis of gene expression,agronomic traits and rice quality.The results showed that no obvious change was observed in mutant bzrl.As to mutants bzr4-1 and bzr4-2,a number of changes were detected,including decreased plant height,reduced length-to-width ratio of flag leaf and the grain number per panicle,increased chalkiness degree,as well as reduced peak viscosity,hot paste viscosity,cool viscosity and the B-type chain content of amylopectin.As to mutants d61-1 and d61-2?decreased plant height and tiller number,increased length-to-width ratio of flag leaf,smaller grain and reduced chalkiness degree were observerd.Moreover,d61-1 and d61-2 showed increased AAC,peak viscosity,hot paste viscosity,cool viscosity and reduced content of low molecular weight amylopectin.2.We created transgenic rice lines of BZR gene family by using the latest CRISPR/Cas9 genome editing technology.Specific and conserved DNA sequences from the exons of four BZR genes were selected as target sites for gene editing to generate mutants with single gene or multiple genes mutated.We successfully obtained a series of rice mutants with single or multiple target genes mutated according to the sequencing results.Moreover,abundant mutation types existed in the mutants.For instances,six(D1,D2,D3,D5,D9 and I1),five(D3,I1,S1,S2 and S3),five(D1?D2?D5,D8 and I1)and two(D4 and D5)kinds of mutation types were included in the single mutant of BZR1,BZR2,BZR3 and BZR4,respectively.And three different combinations of double mutants including BZR1 BZR2,BZR1 BZR3 and BZR2 BZR3,were harvested,with four,seventeen and two kinds of mutation types included,respectively.Finally,the triple mutant BZR1 BZR2 BZR3 had eight mutation types,including D1-S4-D2,D1-I1-D4,D3-D3-D4,D1-D3-D4,D3-D3-I1,I1-D3-I1,I1-S1-I1 and I1-S1-D3.These generated mutants would be helpful to the following function analysis of BZR family genes.3.The BR biosynthesis gene OsDWF4 was cloned into overexpression vector pCAMBIA-Gtl and pCAMBIA-Ubi,respectively.And the generated vectors were transformed into rice cultivar Yandao 8.Then molecular identification,gene expression analysis,agronomic traits investigation and rice quality analysis of the transgenic rice were carried out.The results of molecular identification showed that 46 and 13 independent transgenic lines of pGtl::OsDWF4 and pUbi::OsDWF4 were obtained,respectively.Expression analysis indicated that OsDWF4 expressed higher in the homozygous transgenic lines.The results of agronomic traits analysis indicated that plant height of pGtl::OsDWF4 lines was increased while that of pUbi::OsDWF4 lines was decreased.In addition,the grain length and 1000 grain weight of the transgenic plants overexpressing OsDWF4 increased though other agronomic traits were not changed.Further physicochemical analysis of rice indicated that except RVA profiles,no changes in apparent amylose content and distribution of starch chain lengths were observed in the OsDWF4 transgenic rice.