Modulating Seed Oil Content In Brassica Napus By Interfering With AP2 And Synthesis Of Seed Storage Proteins

Brassica napus is a member of Cruciferae and is one of the most important oilcrops in China and the world.The aim of this study is to improve the oil content in rapeseed through two different ways:1.Since AP2 gene is a negtive regulator of fatty acid biosynthesis,thus repressed expression of AP2 gene may be helpful for improving seed oil content.2.In rapeseed,protein synthesis and oil biosynthesis is negtively correlated,reduced expression of Napin and Cruciferin resulting in a lower storage protein content might lead to an increase in seed oil content.According to above ideas,pCambia 2300-35S-NPT ?-napin promoter-AP2 RNAi construct was introduced into rapeseed;two constructs pCambia 2300-35S-NPT ?-napin-promoter-napin-cruciferin and pCambia 2300-PKA-napin promoter-AP2 RNAi were transformed into repaseed by co-transformation method.The transgenic plants were identified by PCR.We also analysed the difference between transgenic plants and wildtype plants in the expression of AP2,napin and cruciferin genes and the change in thousand seed weight,protein and oil content.These study laid a theoretical foundation for subsequent work.The main results include:1.NPT ? and cAMP-dependent protein kinase A(PKA)gene fragments were used for markers for PCR screening of the transgenic plants containing different plant expression vectors.Transgenic plants harboring pCambia 2300-35S-NPT ?-napin promoter-AP2 RNAi construct with NPT ? marker gene was named as BnAP2a;transgenic plants harboring pCambia 2300-PKA-napin promoter-AP2 RNAi construct with PKA gene and reduced expression of AP2 gene were named as BnAP2b plant;transgenic plants pCambia 2300-35S-NPT ?-napin-promoter-napin-cruciferin RNAi construct with NPT ? marker gene with reduced expression of Napin and cruciferin genes were named as BnNC;transgenic plants pCambia 2300-35S-NPT ?-napin-promoter-napin-cruciferin RNAi and pCambia 2300-PKA-napin promoter-AP2 RNAi constructs with both NPT ? and PKA marker genes and reduced expression of AP2,napin and cruciferin were named as BnANC plant.In 4468 T1 transgenic plants,86 were identified as BnAP2a,146 as BnAP2b,50 as BnNC and 346 as BnANC plant.In 564 T2 transgenic plants,36 were identified as BnAP2a,24 as BnAP2b plant,152 as BnNC plant,3 as BnANC plant.2.Fluorescence quantitative PCRs were performed in wild-type plants at 10DAP,15DAP and 20DAP.It was found that the expression of AP2,Cruciferin and Napin gene expression was highest at 10DAP,15DAP and 20DAP,respectively.3.Fluorescence qRT-PCR in dicated that the expression models of AP2,napin and cruciferin were not significantly different in T1 transgenic plants and wild-type plants however,the expression of AP2,napin and cruciferin in T1 transgenic plants were significantly lower than the wild-type plants.4.Fluorescence qRT-PCR indicated that expression of AP2 gene in two T1 lines were lower than in the wild-type.In AP2a-2-21(NPT),the inhibition of AP2 gene at lOdap,15dap and 20dap was 34.02%,46.06%and 11.67%,respectively.In ANC-6-8(NPT/PKA),the inhibition of AP2 gene at 10dap,15dap and 20dap was 76.2%,65.62%,54.93%,respectively.5.Fluorescence qRT-PCR indicated that expression of napin and cruciferin in four strains were lower than in the wild-type.In NC-1-21(NPT)plant,the inhibition of cruciferin gene at 15dap was 59.4%,the inhibition of napin gene at 20dap was 71.08%;In NC-86-L10(NPT)plant,the inhibition of cruciferin gene at 15dap was 61.39%,and the inhibition of napin gene at 20 dap was 97.18%.In ANC-6-8(NPT/PKA),the inhibition of cruciferin gene at 15dap was 20.44%,and the inhibition of napin gene at 20dap was 28.3%;In ANC-36-8(NPT/PKA)the inhibition of cruciferin gene at 15dap was 40.54%,and the inhibition of napin gene at 20dap was 91.98%.6.In 247 T2 transgenic plants,57 of them(9 BnAP2a,15 BnAP2b,4 BnNC and 29 BnANC)showed an average of 17.38%increase in oil content relative to the wild type;66 of them(20 BnAP2a,14 BnAP2b,11 BnNC and 21 BnANC)showed an average 14.39%decrease in seed storage protein content relative to the wildtype;15 of them(3 BnAP2a,4 BnAP2b,8 BnANC)showed an average 1.38g(36.6%)increase in 1000-seed weight relative to the wild type;30 of them(5 BnAP2a,2 BnAP2b,4 BnNC and 19 BnANC)showed an average 0.52 cm(40.9%)increase in the siliqua length relative to the wild type.7.In T2 transgenic plants the seed oil content was negtively correlated with seed storage protein and positively correlated with 1000-seed weight and beak length.Seed oil content was not significantly correlated with siliqua length or seed number per pod.8.Correlation analysis indicated that reduced expression of AP2 gene may result in an increase in seed oil content and 1000-seed weight and reduced expression of napin and cruciferin genes may lead to an increase in seed oil content but decrease in seed storage protein.9.Some BnAP2 plants display male sterility,shorter siliqua length and darker green leaves compared with the wild-type.