| Watermelon is an important cash crop of melons,fruits and vegetables in the world.The traditional breeding cycle of watermelon is long,and it is difficult to aggregate key traits such as excellent quality and strong stress resistance at the same time.Molecular breeding based on stable genetic transformation technology is an effective means to solve this problem.However,the stable genetic transformation of watermelon has problems such as difficulty in regeneration and serious genotype dependence,which seriously limits the innovation and utilization of molecular breeding technology.In this study,the watermelon growth factor chimeric gene GRF4-GIF1 was cloned,the pGH-GRF-GIF overexpression vector was constructed and the stable genetic transformation was carried out,and it was determined that the GRF4-GIF1 gene could significantly improve the transformation efficiency of watermelon and break the watermelon genotype-dependent barrier.Based on this efficient genetic transformation method,combined with the CRISPR-Cas9 gene editing system,the efficient editing of watermelon ClPDS gene was realized.This system was further used to achieve high expression of the red marker gene RUBY in watermelon,and a new RUBY-labeled watermelon germplasm was created.This study lays a technical foundation for the advancement of the watermelon breeding 3.0 era.The main results are as follows:(1)GRFs and GIFs of wheat,Arabidopsis thaliana and watermelon were analyzed phylogenetically.The results showed that AtGRF5(AT3G13960.1 encoding)had the closest homology relationship with TaGRF4 and AtGIF1(AT5G28640.1 encoding)had the closest homology relationship with TaGIF1 in Arabidopsis.Among watermelons,ClGRF4(Cla97C02G034420.1 encoding)had the highest homology with TaGRF4,and ClGIF1(Cla97C02G042620.1 encoding)had the closest homology relationship to TaGIF1.Identification of protein domains showed that,similar to TaGRF4 protein,ClGRFs protein in watermelon has two conserved domains QLQ and WRC at the N terminus.The ClGRF9(Cla97C05G082580.1 encoding)protein has a second WRC domain at the C-terminus.The N-terminus of ClGIFs in watermelons also have conserved domain SNH.(2)Watermelon ClGRF-GIF chimeric gene can mediate the production of efficient and stable genetic transformation.The overexpression vector of watermelon GRF-GIF chimeric gene was constructed,and the stable genetic transformation of watermelon material ’TC’ was carried out,and the results of measuring its overexpression tissue and seedlings showed that the stable genetic transformation efficiency could be as high as 47.02 %,which was significantly increased by nearly 9 times compared with the control material without transformation.The chimeric gene rClGRF4-GIF1 after the target site of mi R396 on the mutant ClGRF4 gene can further increase the genetic transformation efficiency to 67.27 %,which is a significant increase of about 12 times compared with the control comparison,indicating that the GRF-GIF chimeric gene can mediate the production of efficient and stable genetic transformation efficiency.(3)Watermelon rClGRF-GIF chimeric gene can realize stable genetic transformation independent of watermelon genotype.Another 8 watermelon materials were genetically transformed into control empty vector pGH and pGH-r GRF-GIF,respectively.The results showed that for materials that could not be genetically transformed or could hardly be genetically transformed,including ’M57’,’M20’,’97103’ and ’BJ’,efficient genetic transformation was achieved,and the genetic transformation efficiency was 19.64 %,40.93 %,44.44 % and 48.55 %,respectively.For the originally inefficient materials,including ’YL’,’M08’,’148’ and ’WY’,the genetic transformation efficiency reached 57.35 %,53.57 %,61.84 %and 21.94 %,respectively.Compared with the control group of 6.96 %,3.04 %,1.32 % and1.04 %,the results were significantly increased to 8.24 times,17.62 times,46.85 times and21.10 times,respectively,and it was clear that the rClGRF-GIF chimeric gene can further realize the genotype independent of stable genetic transformation of watermelon.(4)Based on the GRF-GIF efficient transformation system and the integration of CRISPR-Cas9 gene editing system,the efficient editing of ClPDS albino gene in watermelon showed that GRF-GIF chimeric genes can indirectly significantly improve gene editing efficiency.(5)Based on the GRF-GIF efficient transformation system,the overexpression vector of the red marker gene RUBY was constructed,and the stable genetic transformation of watermelon was carried out,and the test results showed that the difficult-to-express RUBY gene was overexpressed in watermelon,and the overexpressed plant was a red transgenic plant. |
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