Study On The Improvement Of Grain Weight Of Brassica Napus L.by Editing BnPPD Gene

Brassica napus L.is the species with the highest grain yield among the three kinds of oil rapeseed（Brassica rapa,Brassica juncea and Brassica napus）.It is widely planted in the middle and lower reaches of the Yangtze River and is an important oil crop in China.However,as a major oil consumer,China is facing a serious shortage of vegetable oil self-sufficiency rate,about 60%of the total consumption needs to be imported.Improving rapeseed yield is a major strategic demand of the country,and high yield has always been one of the important goals of rapeseed genetic improvement.The yield of rape is composed of planting density and yield per plant.The number of effective siliques per plant,the number of siliques per pod and the grain weight are the three components of yield per plant.Among the three yield components,the heritability of the number of effective siliques per plant is low,the heritability of the number of seeds per silique is medium,and the heritability of grain weight is the highest.Therefore,increasing grain weight is one of the effective ways to stably improve the yield of rapeseed plants.one.Grain weight of crops is mainly determined by seed size.So far,few QTLs for rapeseed grain weight have been reported,and their genetic mechanism is unclear.Arabidopsis PPDs（PPD1/2）belong to the TIFY family of proteins and are transcriptional regulators that negatively regulate meristem division by promoting early arrest of meristem or scattered meristem cell proliferation during organ development.Limiting organ growth regulates leaf development,seed growth and germination,hypocotyl elongation,stomatal development and flowering time.Targeted PPD degradation increases Arabidopsis seed size.In this study,a CRISPR/Cas9-PPD dual-target vector was constructed to study the effect of knocking out the PPD gene on the grain weight of B.napus,laying a solid foundation for the improvement of Brassica napus kernel weight.The main results of this study are as follows:1.Using A.thaliana PPD1（AT4G14713）and PPD2（AT4G14720）protein series as Query sequences,four PPD homologous genes were identified by Blast alignment of"Darmor-bzh"genome,which were located on chromosomes A1,A8,C1 and C8,respectively.2.Target 1 was designed on the homologous segment of the second exon of Bn A1PPD and Bn C1PPD（containing PAM sequence NGG）,and target 2 was designed on the homologous segment of the first exon of Bn A8PPD and Bn C8PPD,and a dual target was constructed.Vector CRISPR/Cas9-PPD.YG2009 plants were transformed by Agrobacterium-mediated hypocotyl transformation,and a total of 21 regenerated seedlings were obtained,and 18 positive seedlings were obtained through the identification of hygromycin-resistant primers D3-Hyg F and E3-Hyg R,with a positive rate of 85.7%.The target site sequencing analysis was performed on all positive plants,and it was found that there were 16 plants with target 1 mutation,and the total mutation rate was as high as 88.9%.There are 5 mutants.There were 13 mutant plants of target 2,with a total mutation rate of 72.2%,of which 4 were biallelic homozygous mutants and 8were biallelic heterozygous mutants.3.Single-mutation,partial double-mutation and triple-mutation strains of three genes were isolated in T₁ generation.Compared with wild-type YG2009,the seed diameter of bna1ppd-1,bna8ppd-1,bnc1ppd-1,bnc8ppd-1 and bnc8ppd-1 increased by5.1%,9.9%,17.1%and 19.4%,respectively,all reaching significant levels.The seeds of bna1ppd-1bnc1ppd-1 bipartite plants increased by about 22.3%,which showed an additive effect compared with those of bna1ppd-1 and bnc1ppd-1.The 1000-grain weight of wild YG2009 is 3.561±0.021 g,that of bna1ppd-1 is 3.762±0.033 g,that of bna8ppd-1 is 3.917±0.025 g,that of bnc1ppd-1 is 4.177±0.029 g,and that of bnc8ppd-1 is4.24±0.032 g.The 1000-grain weight of bna1ppd-1bnc1ppd-1 is 4.353±0.027 g,which is additive compared with bna1ppd-1 and bnc1ppd-1.4.Analysis of pod length and quality traits of the offspring of homozygous gene editing:wild type YG2009 pod length is 5.9±0.12 cm,bna1pdd-1 pod length is 6.3±0.12cm,bna8ppd-1 pod length is 6.2±0.22 cm,bnc8ppd-1 pod length is 6.28±0.24 cm,The pod length of bna1ppd-1bnc1ppd-1 is 7.5±0.12 cm,that of bna1ppd-1bnc8ppd-1 is7.7±0.22 cm and that of bna1ppd-1bnc1ppd-1bnc8ppd-1 is 9.7±0.24 cm.Compared with the wild type,the mutant pod length is 7.7±0.24 cm.Wild YG2009 has an oil content of（42.1±0.12）%,bna1ppd-1（43.1±0.14）%,bna8ppd-1（43.5±0.18）%,bnc8ppd-1（43.7±0.21）%and bna1ppd-1ba8ppd-1（44.5±0.22）%.The oil content of bna1ppd-1bnc1ppd-1 is（45.1±0.18）%,and that of bna1ppd-1bnc1ppd-1bnc8ppd-1 is（46.1±0.15）%.Compared with the wild type,the oil content of mutants is significantly increased（p<0.05）.Compared with the wild type,oleic acid,erucic acid,the number of effective pods per plant and the number of seeds per pod had no significant difference.The results of plot yield analysis of the offspring of homozygous gene editing show that under different planting densities,both Bn PPD single mutant and double mutant can increase yield.Under the density of 22,000 plants/mu,the yield per mu of wild YG2009was 172.5±3.22 kg/mu,and that of bna1ppd-1bnc1ppd-1 was 186.1±2.24 kg/mu,with the largest increase of 7.88%.Under the density of 41,000 plants/mu,the yield per mu of wild YG2009 was 177.5±3.56 kg/mu,and that of bnc8ppd-1 was 188.7±3.96 kg/mu,with the largest increase of 6.31%.