Preliminary Analysis Of The Mechanism Of CsLL Gene In Regulating Lateral Branch Development In Cucumber

Cucumber is one of the most important vegetable crops and branch is a key factor determining plant type and yield of cucumber.The branch number of cucumber is a complex character controlled by genetic factors and influenced by hormones and cultivation environment.At present,the research progress on the regulation mechanism of cucumber branch development is slow,which mainly focuses on morphological observation,QTL mapping analysis,homologous cloning and gene mapping.There are very few key genes that have been cloned in cucumber branch regulation,and the biological basis and genetic regulation network that determine the number of cucumber branch are still not clear.Therefore,identification of key genes controlling the number of cucumber branches and analysis of their mechanism are of great theoretical value and practical significance for the improvement of cucumber plant type and breeding of new varieties.A key gene CsLL（KX450264）,which controls organ size and branch number of cucumber,was identified by forward genetic mapping,and a near isogenic line material HAUC106 of ll gene was constructed by marker-assisted breeding.On this basis,we further verified the biological function of CsLL gene in regulating branch number through genetic transformation,and analyzed its genetic regulatory network through yeast hybridization.These results laid a solid foundation for further understanding the molecular mechanism of cucumber CsLL gene.The specific results are as follows:1.Phenotypic investigation was conducted on the number of branches of ll mutant H19,Huabechinotype 9930 and HAUC106 of cucumber planted in the field for 30 days.The results showed that 9930 had 3 branches per plant on average,while ll mutant H19 and HAUC106 had 12 or 9 branches per plant,respectively.These results indicated that CsLL gene was involved in regulating axillary bud differentiation in cucumber.2.In order to verify the biological function of CsLL gene in cucumber,we constructed an overexpression vector of CsLL gene,CsLL-PFGC5941-Flag,which was transformed into cucumber HAUC106 material mediated by Agrobacterium tumefaciens for functional complementary verification.Six positive plants were obtained by screening the resistance of explants,PCR and test paper identification.Compared with the control,CsLL overexpressed plants showed a significant decrease in the number of branches and almost no branches.q RT-PCR results showed that the expression level of CsLL gene in transgenic plants was significantly increased,indicating that CsLL overexpression inhibited the differentiation of lateral branches,suggesting that CsLL is a negative regulatory factor controlling branch of cucumber.In addition,leaf area of positive plants increased and wrinkled,and leaf edge serrated.3.Using CRISPR/Cas9 technology,the target site was designed in the CDS region of CsLL gene by using the online website of gene knockout target design,and connected with p YLCRISPR/Cas9 vector,the gene editing vector of CsLL gene with four target sites was successfully constructed,and the genetic transformation experiment was carried out in cucumber.Two positive tissue culture seedlings with Bar resistance were obtained.However,no editing effect was found by DNA sequencing,and the establishment of this system laid a technical foundation for further knockout and verification of CsLL gene function.4.In order to analyze the expression pattern of CsLL gene in cucumber,in situ hybridization experiments were carried out on axillary buds of 9930 and HAUC106 and oovary at 0 d of flowering.The results showed that the expression signal of CsLL gene in HAUC106 was stronger than that in 9930.Subcellular localization prediction showed that CsLL gene was located in the nucleus,and 35S: CsLL-GFP vector was constructed for instantaneous transformation of tobacco,which also showed localization in the nucleus.5.Preliminary transcriptome analysis found that multiple TCP transcription factors may be involved in the regulatory network of CsLL gene.To further verify whether CsLL gene interacts directly with these TCP transcription factors,we selected6 TCP transcription factors for point-to-point yeast double-hybrid verification.Among them,CsLL could interact with CsTCP7（Csa5G352090）,CsTCP15（Csa1G033030）and CsTCP2（Csa V3₁G004660）,and the interaction relationship was further verified by bimolecular fluorescence complementation experiment.