| As a precursor of vitamin A,carotenoids can scavenge free radicals,prevent cancer,and protect the human’s immune system.Many food crops contain only a small amount of carotenoids,and carotenoids as fat-soluble substances,when simply ingesting staple food crops,the absorption rate is very low,and the absorption of carotenoids by the fats in food has a significant synergistic effect,so increasing the carotenoid content of oil crops can effectively increase the human’s intake of carotenoids.Brassica napus is an important oil crop in China.In the past,the improvement of rapeseed quality was dominated by double low rapeseed with low erucic acid content and low glucosinolates content.However,as people’s needs change,people put forward higher requirements for the quality of rape,To promote the improvement of rapeseed quality in other aspects,so cultivating rapeseed varieties with high carotenoid content has become a new goal for cole breeding.Previous studies have shown that carotenoid cleavage dioxygenases(CCD4)are the main genes involved in the degradation of carotenoids in plants.In Arabidopsis thaliana,GWAS analysis determined that At CCD4 is a key gene that affects the content of carotenoids in seeds.The loss of At CCD4 function can significantly increase the content of carotenoids in seeds.In this study,using Brassica napus as material,three copies of CCD4(Bna A1CCD4,BnaC1CCD4,and Bna A8CCD4)were knocked out simultaneously using CRISPR / Cas9 technology,thereby creating mutant strains with different CCD4 copy combinations for functional differentiation of different CCD4 copies.The research lays the foundation to provide planting resources for the improvement of rapeseed carotenoids.The main findings are as follows:1.Qualitative and quantitative analysis of carotenoids in Brassica napus seeds:High-performance liquid chromatography(high performance liquid chromatography,HPLC)was used to detect the seeds of 17 Brassica napus varieties.The results showed that the carotenoids were mainly lutein and β-carotene.Among these varieties,Hua 5861’s The content of lutein is the highest at 39.27μg / g,while the content of Hua572 is the lowest at 6.02μg / g,a difference of 6.5 times;the content of β-carotene at Hua291 is the highest at 2.21μg / g,while Hua288 is the lowest,0.08μg / g,a difference of 27.6 times,indicating that there is no difference in carotenoid types between different varieties,but there is a significant difference in content.2.BnaCCD4 gene sequence analysis of Brassica napus: Analysis of BnaCCD4 gene sequence in Brassica napus: Through gene sequence analysis,it was found that there are four homologous genes Bna A1CCD4,BnaC1CCD4,Bna A8CCD4 and BnaC3CCD4 in Brassica napus genome,and BnaC3CCD4 has 7.6kb CACTA transposon insertion,Bna A1CCD4 and BnaC1CCD4 The CDS sequence similarity is 97%;the CDS sequence similarity of Bna A8CCD4 and Bna A1CCD4 and BnaC1CCD4 is 86% and 88%,respectively.The similarity between the three copies of the nucleotide sequence indicates that there may be similarities in the functions between them.3.Analysis of BnaCCD4 expression pattern in Brassica napus: The transcription level of three homologous genes of BnaCCD4(Bna A1CCD4,BnaC1CCD4 and Bna A8CCD4)was detected by q PCR in Brassica napus strain A 9707.The results showed that they were expressed in the stems,leaves,flowers and seeds of rape.The expression levels in roots and carobs are low.For seeds of different developmental stages,the expression levels of Bna A1CCD4 and BnaC1CCD4 were lower in the development seeds10 to 50 days after flowering in 10 to 20 days,and the expression levels began to rise from 25 days,and seeds at 50 days after flowering The expression level of the two copies in the medium reached the peak;while the expression level of Bna A8CCD4 decreased from 10 to 25 days after flowering as the seed development period progressed,then increased from 25 to 40 days after flowering with the seed development period,and 50 after flowering Days have begun to decline.There are significant differences in the expression levels of the three copies in different tissues of plants and different developmental stages of seeds.4.Acquisition of BnaCC4 multiple Brassica napus mutant materials: Using gene editing technology,five different specific targets were designed for different copies,and p YLCRISPR / Cas9Pubi-H-9TD and p YLCRISPR / Cas9Pubi-H-9T3 knockouts were constructed respectively In addition to the carrier.The two vectors were transformed into A 9707 by Agrobacterium-mediated method,and 16 p YLCRISPR / Cas9Pubi-H-9TD vector editing plants and 38 p YLCRISPR / Cas9Pubi-H-9T3 vector editing plants were obtained,including three single mutations.Bna A1ccd4,BnaC1ccd4 and Bna A8ccd4,three double mutants Bna A1ccd4 BnaC1ccd4,Bna A1ccd4 Bna A8ccd4,BnaC1ccd4 Bna A8ccd4,and three mutant Bna A1ccd4 BnaC1ccd4.5.Detection of carotenoid content in seeds of BnaCCD4 mutant line :high-performance liquid chromatography(HPLC)was used to detect the content of carotenoid in the seeds of T1 homozygous tribulus lines 208,209 and 29-2.The results indicated that the types of carotenoids in seeds of Jia 9707 and mutant strains mainly include lutein and β-carotene.The content of lutein in the seeds of mutant line 29-2 was97.19μg / g,which was 5.8 times higher than that of Jia 9707;the content of β-carotene was 22.08μg / g,which was higher than that of 9970.27 times;β-cryptoxanthin was detected in the mutant lines at a content of 3.33μg / g.Mutation of three copies of BnaCCD4 in Brassica napus by gene editing technology can significantly increase the content of carotenoids in rape seeds,especially the content of β-carotene. |
