Rapid and efficient isolation of unknown flanking DNA sequences adjacent to known regions is important for molecular biology research.For this purpose,several PCR-based methods have been reported,including inverse PCR...Rapid and efficient isolation of unknown flanking DNA sequences adjacent to known regions is important for molecular biology research.For this purpose,several PCR-based methods have been reported,including inverse PCR(Uchiyama and Watanabe,2006),ligation-mediated PCR(Yan et al.,2003;Ballester et al.,2005;Wang et al.,2007;Trinh et al.,2012)and randomly primed PCR(Liu and Whittier,1995;Liu et al.,1995;Antal et al.展开更多
Carotenoids are important phytonutrients with antioxidant properties,and are widely used in foods and feedstuffs as Supplements.Astaxanthin,a red-colored ketocarotenoid,has strong antioxidant activity and thus can ben...Carotenoids are important phytonutrients with antioxidant properties,and are widely used in foods and feedstuffs as Supplements.Astaxanthin,a red-colored ketocarotenoid,has strong antioxidant activity and thus can benefit human health.However,astaxanthin is not produced in most higher plants.Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes,sZmPSY1,sPaCrtl,sCrBKT,and sHpBHY,which encode the enzymes phytoene synthase,phytoene desaturase,β-carotene ketolase,and β-carotene hydroxylase,respectively.Transgneic overexpression of two (sZmPSY1 and sPaCrtl),three (sZmPSY1,sPaCrtl and sCrBKT),and all these four genes driven by rice endosperm-specific promoters established the Carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm,from the yellow-grained β-caro- tene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice,respectively. Grains Of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity.These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in therice endosperm.This work provides a Successful example for synthetic biology in plants and biofortification in crops;the biofortified rice products generated by this study could be consumed as health-promoting foods and processed tO produce dietary supplements.展开更多
Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice （Oryza sativa L.）, could provide health-promoting foods for improving human health. However, e...Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice （Oryza sativa L.）, could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes （two regulatory genes from maize and six structural genes from Coleus） driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm ＂Purple Endosperm Rice＂ （called ＂Zijingmi＂ in Chinese）, which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation （or enhancement） of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.展开更多
基金supported by grants from the Ministry of Agriculture of China (2016ZX08010001)the National Natural Science Foundation of China (31771740)
文摘Rapid and efficient isolation of unknown flanking DNA sequences adjacent to known regions is important for molecular biology research.For this purpose,several PCR-based methods have been reported,including inverse PCR(Uchiyama and Watanabe,2006),ligation-mediated PCR(Yan et al.,2003;Ballester et al.,2005;Wang et al.,2007;Trinh et al.,2012)and randomly primed PCR(Liu and Whittier,1995;Liu et al.,1995;Antal et al.
基金The National Natural Science Foundation of China (317717,40)Guangdong Province Public Interest Research and Capacity Building Special Fund (2016A020210084+2 种基金2015B020201002)and the Ministry of Agriculture of China (2016ZX080100012016ZX08009002).
文摘Carotenoids are important phytonutrients with antioxidant properties,and are widely used in foods and feedstuffs as Supplements.Astaxanthin,a red-colored ketocarotenoid,has strong antioxidant activity and thus can benefit human health.However,astaxanthin is not produced in most higher plants.Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes,sZmPSY1,sPaCrtl,sCrBKT,and sHpBHY,which encode the enzymes phytoene synthase,phytoene desaturase,β-carotene ketolase,and β-carotene hydroxylase,respectively.Transgneic overexpression of two (sZmPSY1 and sPaCrtl),three (sZmPSY1,sPaCrtl and sCrBKT),and all these four genes driven by rice endosperm-specific promoters established the Carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm,from the yellow-grained β-caro- tene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice,respectively. Grains Of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity.These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in therice endosperm.This work provides a Successful example for synthetic biology in plants and biofortification in crops;the biofortified rice products generated by this study could be consumed as health-promoting foods and processed tO produce dietary supplements.
基金This work was supported by grants from National Natural Science Foundation of China （31000698）, the Ministry of Agriculture of China （2016ZX08010001 2016ZX08009002+1 种基金 2014ZX08010001）, and Guangdong Province Public Interest Research and Capacity Building Special Fund （2015B020201002 2016A020210084）.
文摘Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice （Oryza sativa L.）, could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes （two regulatory genes from maize and six structural genes from Coleus） driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm ＂Purple Endosperm Rice＂ （called ＂Zijingmi＂ in Chinese）, which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation （or enhancement） of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.
