Enhancing carotenoid biosynthesis in rice endosperm by metabolic engineering

First author: Yong‐Sheng; Affiliations: Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences (上海农科院生物技术研究所): Shanghai, China

Corresponding author: Quan‐Hong Yao

Most cereal grains contain small amounts of dietary carotenoids. Thus, vitamin A deficiency (VAD) is common in third‐world countries where the nutrition (of the population) depends on a single staple crop such as rice. Thus, considerable research effort has been dedicated to achieving sustainable and economical production of carotenoids in crops (Paine et al., 2012). Plant carotenoids are isoprenoid‐derived molecules, and isoprenoids are produced via the mevalonate (MVA) or plastidial 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) routes, both giving rise to isopentenyl diphosphate (IPP) (Hemmerlin et al., 2012). However, little is known about the influence of the genes involved in the MVA or MEP pathways on carotenoid production in transgenic plants.

大多数禾本科植物的籽粒含有的能够食用的类胡萝卜素物质比较少。因此，维生素A缺乏病VAD在单一依赖水稻等谷类作物为主食的第三世界国家比较常见。因此，全球范围很多研究人员投入到能够稳定、经济提升作物中的类胡萝卜素的研究中。植物中的类胡萝卜素是异戊二烯衍生物，而异戊二烯类物质是由甲羟戊酸途径MVA或质体中的2-C-甲基-D-赤藻糖醇-4-磷酸MEP途径所产生的，这两个通路都能够生成异戊烯焦磷酸IPP。然而，关于MVA或MEP通路上相关基因在转基因植株中的类胡萝卜素的产量方面的作用还所知不多。

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is generally considered the major rate-limiting enzyme in the MVA pathway. Transgenic tomatoes over-expressing plant HMGR without also over-expressing the genes encoding phytoene synthase (Psy) and phytoene desaturase (CrtI) show IPP accumulation and no carotenoid (over-) accumulation (Enfissi et al., 2005). To further investigate the effect of HMGR (overexpression) on carotenoid pathways in rice endosperm, we heterologously expressed Psyfrom maize (ZmPsy) and CrtIfrom Pantoea ananatis (PaCrtI) along with tHMG1, which encodes truncated HMGR from Saccharomyces cerevisiae, in this study. The three genes (tHMG1,ZmPsy1and PaCrtI1) were chemically synthesized through PCR-based two-step DNA synthesis (PTDS) (Xiong et al., 2006). The codons of these genes were optimized and preferentially designed for rice. Then, a gene expression cassette (GtHMG1, GZmPsy1andGPaCrtI1) was constructed by inserting the ORF between an endosperm-specific rice globulin-1 promoter and terminator using the PAGE-mediated overlap extension PCR method (Peng et al., 2014). In this way, different restriction sites were added to the 5’ and 3’ ends of the expression cassette. The starting point for the construction of the plasmid for rice transformation was pCAMBIA-1301, and the vector was modified through the introduction of new multiple cloning sites to yield the plant binary vector pYP694 (Peng et al., 2014). Then, pYP69-GtHMG1was prepared by inserting the GtHMG1 expression cassette into pYP694 at the Eco RI and Bam HI sites, and GZmPsy1was inserted into the pYP69-GtHMG1vector at the Bam HI and Kpn I sites to obtain the two-gene construct pYP69-GtHMG1-GZmPsy1. Finally, the three-gene construct (pYP69-GtHMG1-GZmPsy1-GPaCrtI1, genotype HPC) was obtained using the Kpn I and Xba I sites of the above mentioned two-gene construct and named pYR2420. (Figure 1a).  

3-羟基-3-甲基戊二酰辅酶A还原酶HMGR是MVA通路的限速酶。单独过表达HMGR而不过表达八氢番茄红素合成酶Psy基因和八氢番茄红素脱氢酶CrtI基因的番茄植株中，IPP的积累增加，但类胡萝卜素含量并不会显著增加。为了进一步验证在水稻胚乳中过表达HMGR对于类胡萝卜素的影响，作者将截短了的酿酒酵母HMGR编码基因tHMG1和玉米的ZmPsy基因、菠萝泛菌的PaCrtI基因一起在水稻中异源表达。作者通过基于PCR的两步DNA合成法PTDS化学合成了这三个基因。作者通过基于水稻的最优密码子设计重新设计了这三个基因的核苷酸序列。最终，作者通过将GtHMG1、GZmPsy1和GPaCrtI1插入到一个水稻胚乳特异性表达球蛋白-1的启动子和PAGE介导的重叠延伸PCR技术形成的终止子中间，形成了一个表达组件。通过这种方法，作者在该表达载体的5’和3’端添加了不同的酶切位点。作者利用pCAMBIA-1301载体做为水稻转基因的构件骨架，并通过引入新的克隆位点进一步修饰该载体以形成植物双向表达载体pYP694。然后，作者通过Eco RI和Bam HI酶切位点将GtHMG1表达组件插入到pYP694，并通过Bam HI和Kpn I酶切位点将GZmPsy1插入到pYP69-GtHMG1载体上以获得包含两个基因的表达载体pYP69-GtHMG1-GZmPsy1。最终，作者通过Kpn I和Xba I酶切位点将GPaCrtI1插入到pYP69-GtHMG1-GZmPsy1载体上形成了三个基因的表达载体pYP69-GtHMG1-GZmPsy1-GPaCrtI1。

doi: https://doi.org/10.1111/pbi.13059

Journal: Plant Biotechnology Journal

First Published: 24 December, 2018