Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in‐depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.     

Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a beta-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)-selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.     

Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

水稻sbe1启动子驱动的反义sbe-GUS融合基因在转基因水稻中的表达

为研究水稻基因启动子对外源基因在转基因水稻中表达的影响,构建了由sbe1启动子引导的反义sbe-GUS融合基因。经农杆菌介导,将不同的融合基因导入水稻中,定量测定转基因水稻植株不同组织中的GUS酶活力。结果表明,sbe1启动子可驱动反义sbe-GUS融合基因在转基因水稻植株的胚乳中高效表达,而在颖壳、胚和茎叶等组织中的表达活性较弱。证实sbe1启动子在驱动外源基因的表达上表现有明显的组织特异性。     

农杆菌介导的玉米遗传转化

Several maize inbreds were transformed with Agrobacterium tumefaciens EHA101 (pGIH). Transgenic maize plants were obtained. Frequency of transformation of maize inbred Suyu No. 1 can reach 8.1%. Results of PCR and Southern blot analysis proved that T-DNA was stably integrated into the genome of maize. Staining with X-gluc confirmed the expression of GUS gene in maize cells. The band amplified by inverse PCR showed that the copy number of transgene in three transformants was single. After long term of subculture, some hygromycin resistant calli lost their regeneration ability. Although Southern blot probed the integration of gusA gene in their genome, GUS activity cannot be detected in those calli. Southern blot analysis of HpaII digest DNA showed that transgenic gusA gene was highly methylated.     

Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.     

Expression pattern and activity of six glutelin gene promoters in transgenic rice

The shortage of strong endosperm-specific expression promoters for driving the expression of recombinant protein genes in cereal endosperm is a major limitation in obtaining the required level and pattern of expression. Six promoters of seed storage glutelin genes (GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC) were isolated from rice (Oryza sativa L.) genomic DNA by PCR. Their spatial and temporal expression patterns and expression potential in stable transgenic rice plants were examined with beta-glucuronidase (GUS) used as a reporter gene. All the promoters showed the expected spatial expression within the endosperm. The GluA-1, GluA-2, and GluA-3 promoters directed GUS expression mainly in the outer portion (peripheral region) of the endosperm. The GluB-5 and GluC promoters directed GUS expression in the whole endosperm, with the latter expressed almost evenly throughout the whole endosperm, a feature different from that of other rice glutelin gene promoters. The GluB-3 promoter directed GUS expression solely in aleurone and subaleurone layers. Promoter activities examined during seed maturation showed that the GluC promoter had much higher activity than the other promoters. These promoters are ideal candidates for achieving gene expression for multiple purposes in monocot endosperm but avoid promoter homology-based gene silencing. The GluC promoter did not contain the endosperm specificity-determining motifs GCN4, AACA, and the prolamin-box, which suggests the existence of additional regulatory mechanism in determining endosperm specificity.     

采用玉米Ubi-1启动子获得低拷贝转基因玉米植株

通过基因枪粒子轰击和草丁膦（PPT）选择获得可育的玉米转基因植株,并分析了外源基因在转化体中的拷贝数与启动子之间的关系。用玉米Ubi-1启动子驱动外源基因,玉米转化体中外源基因的拷贝数较低;可能的原因为Ubi-1启动子通过与其内部同源序列发生重组而被定点整合进玉米基因组,共转化的两种质粒DNA在整合至玉米染色体DNA之前已重构成为一个整体。结果显示使用某一植物自身基因的启动子可以降低外源基因在该物种转基因个体中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因玉米种子。     

Promoters of Rice Seed Storage Protein Genes Direct Endosperm-Specific Gene Expression in Transgenic Rice

The promoters of genes encoding rice seed storage proteins (glutelin,prolamin, globulin and albumin) were analyzed for their abilityto direct rß-gIucuronidase (GUS) gene expression intransgenic rice plants. All promoters tested could direct endosperm-specificexpression of the GUS reporter gene irrespective of variableactivities and patterns in the endosperm. (Received February 27, 1998; Accepted May 16, 1998)     

Enhanced expression of phospholipase C 1 (ZmPLC1) improves drought tolerance in transgenic maize

Phosphatidylinositol-specific phospholipase C (PI-PLC) plays an important role in a variety of physiological processes in plants, including drought tolerance. It has been reported that the ZmPLC1 gene cloned from maize (Zea mays L.) encoded a PI-PLC and up-regulated the expression in maize roots under dehydration conditions (Zhai SM, Sui ZH, Yang AF, Zhang JR in Biotechnol Lett 27:799–804, 2005). In this paper, transgenic maize expressing ZmPLC1 transgenes in sense or antisense orientation were generated by Agrobacterium-mediated transformation and confirmed by Southern blot analysis. High-level expression of the transgene was confirmed by real-time RT-PCR and PI-PLC activity assay. The tolerance to drought stress (DS) of the homogenous transgenic maize plants was investigated at two developmental stages. The results demonstrated that, under DS conditions, the sense transgenic plants had higher relative water content, better osmotic adjustment, increased photosynthesis rates, lower percentage of ion leakage and less lipid membrane peroxidation, higher grain yield than the WT; whereas those expressing the antisense transgene exhibited inferior characters compared with the WT. It was concluded that enhanced expression of sense ZmPLC1 improved the drought tolerance of maize.     

水稻谷蛋白GluA-2基因5′上游序列控制下的UidA基因在转基因水稻胚乳中的表达模式

为了研究谷蛋白胚乳特异性表达启动子在我国栽培稻品种中的表达模式,将UidA基因分别置于水稻谷蛋白GluA—2基因750bp和2．3kb上游序列下游,利用农杆菌转化法导人栽培稻品种中花8号并获得转基因植株。Southern blot检测表明,UidA基因已经整合到水稻基因组当中并以单拷贝存在。Northern blot检测表明,开花后13～15d和11～13d,UidA基因和水稻内源的GluA—2基因的表达量分别达到最高,随后逐渐降低。对转基因植株种子的GUS染色表明,UidA基因仅在胚乳中表达,在糊粉层中GUS表达量最高。测定了2．3kb和750bp转基因植株种子的GUS活性,结果表明前者的GUS活性是后者的2～3倍。序列分析表明,位于GluA—2基因转录启始位点上游2170bD的G-box可能是一个与表达量相关的顺式调控元件。