The Effect Of Different Transgenic Constructs On Regulation Of Amylose Content In The Endosperm Of Rice (Oryza Sativa L.)

Amylose content is one of the key factors determining the grain quality in rice, and it can be altered by transgenic regulation of the expression of starch synthesis-related genes, especially the Waxy (Wx) gene. In present study, several approaches were applied to regulate the amylose content in rice endosperm, including the down-regulation of the Waxy (Wx) expression via antisense RNA approach, up-regulation of Wx expression via over-expression approach, down–regulation of the expression of starch branching enzyme genes Sbe1 and/or Sbe3 via RNA interference approach, and the combination of the above approaches. The homozygous transgenic rice, some of them without the selectable marker, were subsequently selected from lots of primary transformants, and the grain quality and its related characters were carefully investigated and shown as followings:1. The amylose synthesis in rice endosperm could be down regulated by antisene inhibition the expression of rice endogenous Wx gene. In our previous study, lots of co-transgenic rice plants, containing both the antisense-Wx gene and selectable marker (hygromycin resistance gene, HPT), had been generated through Agrobactium-mediated co-tranformation system. In this study, from the offsprings of the co-transformants derived from the cultivars Wuxiangjing 9, Xieqingzao and Longtefu, we selected and identified several selectable marker-free (SMF) transgenic rice containing the homozygous antisense-Wx gene, and the grain quality in the mature seeds of these selected lines were then measured. The amylose content in mature seeds of these SMF transgenic rice were reduced to different level when compared to that of their wild type, and it has reached the level of the waxy rice (below 2%) in several lines. The expression analysis indicated that the expression of endogenous Wx gene (the amount of GBSS) is down-regualted in the endosperm of transgenic rice plants with decreased amylose content. When the amylose content was reduced in mature seeds of transgenic rice, the gel consistency became more soften when comppared with that of the wild type, and some of the RVA characteristics were also changed. But the effect of the decreased AC on the gelatinization temperature was different among various transgenic lines derived from different varieties. The down-regualtion of AC has a significant effect on the structure of the starch granule, observed by scanning electron microscope, in mature seeds of the transgenic plants. The results from field trials showed that the rice grain weight of the transgenic rice obviously reduced as its amylose content down to very low level and its size also became smaller. But, the other agronomic traits showed no significant alteration in transgenic rice lines when compared with those of the wild type.2. It had been proven that the expression of endogenous Wx gene also could be regulated (co-suppression or over-expression) by introduction of its sense gene. We had generated some transgenic rice plants containing the sense Wx gene (the genomic DNA containing the Wx promoter and its full-length coding sequences) in different rice cultivars. Among the successive generations via self-pollination of these transformants, 30 transgenic lines containing the homozygous sense Wx gene were subsequently selected, and their grain quality and field performance were investigated, and the results showed that: (1) In most of the transgenic rice lines selected, the amylose content increased to a different level when compared to that of their wild type, except one transgenic line derived from Xieqingzao, in which the amylose content decreased. (2) In most of transgenic rice plants with increased amylose content, the expression of Wx gene (amount of GBSS) was up-regulated, in particular in japonica and waxy rice with low expression level of native Wx gene in their wild type. (3) In transgenic rice plants derived from japonica or waxy rice varieties, when the amylose content increased, the gel consistency became harder. While no change was observed in indica rice. The RVA profile of the starch from transgenic rice also changed, but the change level differs among the transgenic rice derived from different varieties. (4) Of the transgenic rice derived from japonica or waxy rice, the increased amylose had a positive effect on the grain size, i.e, the transgenic brown rice weight was more heavy than that of their wild type.3. The third approach in present study used to regulate the amylose content in rice is to introduce (or co-introduce) the Wx over-expression (full-length Wx-cDNA controlled by the rice glutelin Gt1 promoter) construct and/or the antisense or RNAi structure of Sbe gene(s). Three japonica rice varieties, Wuxiang 9915, Guanglingxiangnuo and Suyunuo, were used for co-transformation mediated with the Agrobacterium, and lots of transgenic plants were regenerated. The results from PCR and Southern blot analyses showed that the target genes had co-integrated, togather with the selectable marker gene, into the genome of rice plants after co-transformation. Thus, among the offspring after self-pollination, several selectabel marker-free transgenic rice plants could be obtained. (2)The amylose content of the mature seeds in T1 generation of some transgenic rice plants increased with a various range when compared to that of the wild type. These transgenic rice plants were very useful for further study to select novel transgenic rice lines with different starch quality.