| Brassica napus L.is one of the most important oil crops in China.In 2022,the production of Brassica napus L.in China is about 14.7 million tons,accounting for 17.5%of the Brassica napus L.production in the world.The SBP-box family is a family of transcription factors unique to plants,and its family members play an important role in various stages of plant growth and development,but there is less research in Brassica napus L..In this study,we created a library of Brassica napus L.SBP-box family mutants by gene editing technology,and preliminarily analyzed the phenotypes of BnaSPL4 and BnaSPL14mutants,and the relevant results are as follows:1.Creation of Brassica napus L.SBP-box family mutant library.There are 59members of the SBP-box family of library,which is named BnaSPL1-BnaSPL16 based on sequence homology to the Arabidopsis SBP-box family.Taking these SPL genes as targets,sg RNAs were designed in the sequence conserved region of homologous copy genes,15CRISPR/Cas 9 vectors were constructed independently and transformed Agrobacterium respectively,and a total of 227 independent transgenic strains were obtained by mixing the hypocotyl of Brassica napus L.with two combinations.In order to identify the vectors transferred in transgenic plants,universal primers were designed before and after vector sg RNAs,PCR products containing sg RNA were amplified,and the high-throughput sequencing results showed that the positive rate of vector transformation was 88.55%,of which plants transferred to a single vector accounted for 55.72%of the positive plants,and plants transferred to multiple vectors accounted for 44.28%of the positive plants.The results showed that the plants with gene editing accounted for 46.77%of the total number of positive plants,of which 86.17%were single SPL gene edited plants and 13.83%were multiple SPL gene editing plants.At the same time,through BnaSPL4 haplotype analysis,it was found that the difference of BnaSPL4 near the 3’end of isoleucine in different varieties was closely related to the difference in flowering time of winter,semi-winter and spring rape.2.BnaSPL4 and BnaSPL14 regulate flowering and branching number in Brassica napus L.,respectively.The function of some members of the SBP-box gene family has been validated in multiple species.Through genotypic analysis of the mutant library,we identified bnaspl4 mutants,bnaspl14 mutants,and bnaspl4 and bnaspl14 double mutants in T1 generation materials.The tissue expression pattern results showed that BnaSPL4 was highly expressed in flower buds and ovary and BnaSPL14 was highly expressed in stems and ovary.The phenotypic analysis results showed that the initial flowering time of bnaspl4mutants was earlier than that of wild type,and the expression of BnaFLC,a negative regulator of flowering,was significantly downregulated in bnaspl4 mutants,and it was speculated that BnaSPL4 may be involved in the regulatory pathways related to the flowering period of Brassica napus L.,while other agronomic traits such as silique length and number if seeds per silique had no significant effect.The bnaspl14 mutant showed a significant phenotype of increasing the number of branches,and its branch number was mainly reflected in the increase of the number of tertiary branches,while the difference between the number of primary branches and the number of secondary branches was not obvious,and it was found that the number of siliques per plant increased significantly,but there was no significant difference in the length of silique,the number of seeds per silique and the weight of 1,000 grains,and the quantitative detection results showed that the expression of branching-negative gene BnaBRC1,BnaHB21 and BnaHB40 in the mutants decreased significantly.It is speculated that BnaSPL14 controls branching by regulating the expression of BnaBRC1,BnaHB21 and BnaHB40.The results of double luciferase reporter gene analysis（LUC）showed that BnaSPL14 had a transcriptional activation effect on BnaBRC1,combined with the high expression of BnaSPL14 in stems,BnaSPL14 may play an important role in the breeding of Brassica napus L.,it may be an important gene for regulating the ideal plant type of Brassica napus L..The bnaspl4/14 double mutant showed obvious phenotypes of early flowering,increased number of branches and increased number of siliques per plant,and the expression trend of BnaFLC,BnaBRC1 and other genes was the same as that of single mutant,and it was further found that the length of silique,the number of seeds per silique and the weight of 1,000 grains decreased significantly,indicating that the loss of BnaSPL4and BnaSPL14 gene functions may affect the normal development of Brassica napus L..Brassica napus L.is an important oil crop,and mining and analyzing the gene function of it is an important part.In this study,a mutant library of Brassica napus L.SBP-box gene family was created,and the flowering stage and branching traits of BnaSPL4 and BnaSPL14mutants were preliminarily investigated.This study lays a foundation for the analysis of the functions of SBP-box gene family members,and provides ideas and candidate genes for revealing the molecular basis of Brassica napus L.yield and adaptability improvement. |
