Study On Molecular Mechanism Of BnaARF2 Regulating Seed Size Of Brassica Napus L.

Seed size is one of the main components of crop yield,and research on seed size has made great progress.In the study of model plants such as Arabidopsis thaliana and rice,various pathways are involved in the regulation of seed size.Auxin response facto r ARF2?Auxin response factor 2?participates in various plant physiological processes,and also plays an important role in the regulation of seed size.There have been relevant reports on ARF2 regulating seed size in Arabidopsis.We successfully created a mutant bnaarf2 of Brassica napus by CRISPR/Cas9 gene editing technology.The seed size of the quadruple mutants L35 and L37 was increased significantly,which was increased by 22%and 27%,respectively.At the same time,yeast two-hybrid and other experiments verified that BnaARF2 may regulate the seed size of Brassica napus seeds by interacting with Bna MPK3.The main experimental results are summarized as follows:There were four paralogues of BnaARF2 in Brassica napus,named Bna A06.ARF2,Bna A09ARF2,Bna C03.ARF2,Bna C09.ARF2.These four paralogues had very high homology,among which the sequence similarity between Bna A06.ARF2 and Bna C03.ARF2 was 97%;the sequence similarity between Bna C03.ARF2 and Bna A09ARF2was 85%.The BnaARF2 protein had three conserved domains,namely the B3 domain at the N-terminus,the Auxin?resp domain at the middle part,and the AUX?IAA domain at the C-terminus.RNA-seq data and q RT-PCR results of various tissues of Brassica napus and various stages of seed development showed that BnaARF2 was expressed in various tissues of Brassica napus,Some paralogues were expressed in specific tissues with higher expression levels,among which Bna C03.ARF2 was highly expressed in cotyledons,Bna C09.ARF2 was highly expressed in flowers and seeds,Bna A09.ARF2 was highly expressed in cotyledons,and Bna A06.ARF2 was highly expressed in stems.The expression level of BnaARF2 gradually decreased during the seed development.BnaARF2 mutants were obtained by CRISPR/Cas9 genome editing techniques.In T₁and T₂ generation the organ sizes of the bnaarf2 quadruple mutants L35 and L37 were significantly increased,and the organ sizes of the triple mutant L38 and double mutant L39were almost same as wildtype.There were no significant defect of other important agronomic traits such as the number of grains per silique and oil content.The measurement of the length and width of the silique revealed that the width of the silique of the bnaarf2?L35 and L37?increased significantly,which was about 37%larger than that of the wild type.The size of mesophyll cells of L35 and L37 were significantly larger than the wild-type materials.The organ size was significantly decreased in Bna C03.ARF2 overexpression lines.The seed area of L4 and L5 was about 28%and 15%smaller than that of wild-type respectively.At the same stage,the size of mesophyll cells of L4 and L5 also decreased significantly.This indicated that Bna C03.ARF2 negatively regulated the seed size in Brassica napus.The results of yeast two-hybrid,luciferase complementation and Pull Down experiments verified the interaction between Bna MPK3 and BnaARF2.The bnampk3 mutant was constructed using CRISPR/Cas9 technology,and two homozygous lines L4 and L5 were identified.The seed size of these two lines was significantly smaller than that of wild-type lines,in which the thousand-grain weight of L4 was reduced by 27%and the thousand-grain weight of L5 was reduced by 29%.In addition,the fresh weight of seedlings of L4transgenic lines was reduced by 60%compared with wild type,and the fresh weight of seedlings of L5 transgenic lines was reduced by 63%compared with wild type.It is preliminary indicated that Bna MPK3 may be involved in the regulation of seed size and biomass of Brassica napus.