Multi-gene Engineering Boosts Oil Content In Rice Grains

Vegetable oils are important sources of nutrients and energy in the human diet.In addition,vegetable oils play a significant role in the construction of the national economy,which are major raw materials for industrial production and renewable “green energy”.Since the 21st century,the demand for edible vegetable oils and bioenergy has boomingly increased in China,resulting in an enormous reliance on the import of vegetable oil,such as soybeans and rapeseed,which has become the urgent "stuck neck" problem in industrial structure of China’s agricultural.Compared with oil crops such as soybeans,rice has the characteristics of high yield but low oil content.Therebefore,the high-oil rice germplasm not only solves the current situation of serious shortage of vegetable oils supply in China,but also improve the commercial added value of rice.Atthe same time,it also can reduce the reliance on the import of oil crops such as soybeans,and ensure national food security in China.In this study,we used synthetic biology methods to simultaneously inhibit the allocation of carbon sources to starch synthesis,enhance oil synthesis in rice seeds,and increase the size of oil storage tissue in a major cultivar variety Nan Geng46(Oryza sativa L.cv.NG46).Finally,the goal of increasing oil storage in rice seeds is ultimately achieved.The research results are as follows:1.Diacylglycerolacyltransferase(DGAT)is the rate-limiting enzyme of the Kennedy reaction that catalyzes diacylglycerol to generate triacylglycerols(TAGs).Heterologous expression of AtDGAT1 in rice endosperm can improve the efficiency of assembling TAGs,which increased the relative or absolute oil content in brown rice by 47.6% and in per brown seed by 14.3%.while starch and soluble sugar content decreased dramatically in AtDGAT1,respectively.Compared to the wild type,AtDGAT1 transgenic plants showed a phenotype with delayed heading date and no changes in other agronomic traits.It can be concluded that the assembly efficiency of TAGs in rice seeds was improved and the oil content of brown rice also was increased by introducing key enzymes to promote oil synthesis in oil plants.2.ADP-glucose pyrophosphorylase large subunit 2(AGPL2)is a rate-limiting enzyme of starch synthesis that catalyzes glucose-1-phosphate(G-1-P)to generate the starch substrate ADP glucose(ADPG).Knocking out AGPL2 gene could increase the relative or absolute oil content in brown rice by4.0 times and in per brown seed by 1.82 times.The soluble sugar and protein content significantly increased while the starch content decreased.Compared with the wild type,agpl2 exhibited increased plant height,delayed heading stage,shrunk seeds,small grain size and decreased 1000-seed weight.It was evidenced that the diverting the flux of carbon from starch to lipids can significantly enhance the oil content.3.In AtDGAT1 background,we knocked out AGPL2 gene to get AtDGAT1/agpl2 transgenic plants.The relative oil content of AtDGAT1/agpl2 brown rice was increased by 4.35 times,the absolute oil content of per brown seed increased by 1.5 times.The soluble sugar and protein content increased while the starch content significantly decreased.Compared with the wild type,AtDGAT1/agpl2 exhibits phenotypes such as delayed heading date,shrunk seeds,small seed size,reduced seed setting rate,and decreased 1000-seed weight.The above results indicated that the strategy combining changing carbon source allocation with enhancing lipid synthesis pathways provides the sight of increasing the lipid content.4.In AtDGAT1 background,we knocked out mitochondrion-targeted single-stranded DNA binding protein(MTSSB1)and AGPL2 to get AtDGAT1/agpl2/mtssb1 transgenic plants.The relative oil content of AtDGAT1/agpl2/mtssb1 brown rice increased by 5.03 folds,the absolute oil content of per brown seed increased by 1.98 folds.The soluble sugar and protein content increased while the starch content significantly decreased.Compared with the wild type,AtDGAT1/agpl2/mtssb1 showed the same agronomic phenotype as AtDGAT1/agpl2.The above results indicated that the lipid content of 11.72%has been very close to oil crops like soybean,which represented the highest lipid level that has ever been reported in the grains of rice or other starchy type grains.5.The identification of the fatty acid components of brown rice in NG46,AtDGAT1,AtDGAT1/agpl2 and AtDGAT1/agpl2/mtssb1 by gas chromatography showed that the saturated fatty acids such as C16:0 and C18:0 increased,while the content of unsaturated fatty acids such as C18:1n9c and C18:2 decreased in brown rice of AtDGAT1,AtDGAT1/agpl2 and AtDGAT1/agpl2/mtssb1.It indicated that AtDGAT1,AGPL2 and MTSSB1 regulated the composition of fatty acids,and provided a new sight for analysis of fatty acid composition in rice.Atthe same time,the increase of C18:0 content in each transgenic brown rice can make rice oil to be used in artificial fat foods while avoiding the formation of trans fatty acids,which provides a new opportunity for increasing the added value of rice.In this study,a high-oil rice germplasm resource AtDGAT1/agpl2/mtssb1 with 11.72% oil content in brown rice was created by regulating carbon source allocation and promoting the lipid synthesis pathway.This research is conducive to improving self-sufficiency rate of edible oil,allowing the “oil bottle” to contain as much Chinese oil as possible.Atthe same time,it could increase the commercial added value of rice and expand the scope of rice utilization and make rice not only a staple food,but also some important raw materials for health care products,food industry and bioenergy.Our results showed diverting the flux of carbon from starch to oil could enhance the oil content in rice seed.This work provides a practical approach for the genetic improvement of oil contents in rice and other crops with starchy grains.