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Long-te-fu (LTF) and Zhan-shan 97 (ZS) are two key female parents for the generation of indica hybrid rice, which have greatly contributed to the achievement of rice production in China. However, the high amylose content (AC) in the endosperm, controlled by the Waxy (Wx) gene encoded granule-bound starch synthase I, of both lines results in poor cooking and eating quality of the milled rice. Our previous studies have shown that AC was correlated with the ability to excise intron 1 from the leader sequence of the Wx transcript, and which is responsible by a single nucleotide polymorphism (G or T) located at the first nucleotide of the splice donor site of Wx intron 1. Thus, a CAPS marker was subsequently developed, and with this molecular marker-assisted selection (MAS) we successfully introgressed the Wx-TT locus of rice cultivars with good quality intermediate AC into LTF-B and ZS-B. These were subsequently introduced into their relevant male-sterile lines (LTF-A and ZS-A) to generate improved indica hybrids. In the selected lines LTF(tt)-B and ZS(tt)-B, the AC was reduced to a relatively low level (15%). Consequently, the hybrids crossed from the selected lines had dramatically reduced amylose levels. In the field trials, the agronomic performance in the improved lines and their hybrids was examined and compared to the originals. The other key factors involved in rice cooking and eating quality were also improved in the selected lines and their hybrids.

Summary: We have previously reported that three distinct patterns of waxy (Wx) gene transcript accumulation were present in 31 rice cultivars. The cultivars with high amylose content (group I) contain a 2.3 kb mature Wx mRNA, cultivars with intermediate amylose content (group II) produce both a 3.3 kb Wx pre-mRNA, which contains intron 1, and the 2.3 kb Wx mature mRNA, and cultivars with no amylose (group III) accumulate only the 3.3 kb Wx pre-mRNA. Analyses of the cDNAs reveals that four splice donor sites and three splice acceptor sites in intron 1 give rise to six splicing patterns in 2.3 kb Wx mRNA of group II cultivars. In addition, aberrant intron 1 excision causes either deletion of 4 or 5 nucleotides, or addition of 7 and 13 nucleotides at the junction of exon 1 and exon 2 of the 2.3 kb mRNA. In contrast, only one normal splicing pattern (one splice donor site and one splice acceptor site) was found in the 2.3 kb mRNA of group I cultivars. Nucleotide sequences of the Wx intron 1 in group I and group II cultivars differ by 16 individual bases. We suggest that these deletions or additions contribute to inefficient splicing of intron 1 from the 3.3 kb Wx pre-mRNA, as well as an aberrant splicing of the Wx intron 1 to produce the 2.3 kb mRNA with a heterogeneous 5′ untranslated region (5′-UTR). As a consequence, the total amount of translatable Wx mRNA, and therefore the Wx protein and amylose content, are reduced in the group II cultivars compared with the group I cultivars. [ABSTRACT FROM AUTHOR]

Abstract: In rice grains, high amylose content (AC) is correlated with poor grain quality, particularly in indica hybrid rice. To obtain indica hybrid rice with improved cooking and eating qualities, we introduced the antisense Waxy (Wx) gene into 2 elite parental lines of indica hybrid rice by using co-transformation methods. Subsequently, we selected several elite homozygous transgenic lines that did not contain the selectable marker. The expression of the endogenous Wx gene of the selected transgenic lines was significantly downregulated, resulting in low AC in the mature seeds; moreover, the AC in some lines reduced to the level observed in glutinous rice. With the decrease in AC, the gel consistency of the transgenic rice became softer, and the gelatinization temperature tended to be higher than those of the wild types, especially in the case of the Longtefu-derived transformants. We also analyzed the pasting properties of the selected transgenic low-AC lines, and we noted an improvement in the pasting properties of the transgenic rice lines. The results from a field trial indicated that the grain weights of the transgenic lines with lower AC exhibit remarkable reduction compared with those of the wild types. [Copyright &y& Elsevier]

Previous data showed that a 31-bp (from -840 bp to -810 bp) DNA fragment located at the 5' upstream region of rice waxy gene could interact with nuclear protein extracted from developing endosperm of rice. When this 31 bp DNA sequence was used as a bait to screen a rice cDNA library with a yeast one-hybrid system, three groups of cDNA clones were isolated. One of them is pC73, the correspondent rice gene of pC73 was named as OsBP-73 (Oryza sativa binding protein). A pull-down assay was made to identify the target genes of transcription factor by using genomic DNA and recombinant p73 protein. The cDNA fragment containing DNA-binding domain of OsBP-73 was cloned into expression vector pET28-c(+) (Fig.1) to produce protein p73, fused with a his(6)-tag, from E. coli BL21 (DE3) (Fig.2). The p73 was purified with Ni-NTA under native condition (Fig.3). The target genes of p73 were identified in rice genome-wide by using a pull-down assay, and 22 candidate genes were obtained (Figs.4 and 5, and Table 1). The obtained results show that putative light-repressible receptor protein kinase and GAMYB-binding protein could serve as targets of the OsBP-73, suggesting that OsBP-73 might be involved in light signal transduction.

The coding region of Bar gene, the left border of Ds element, the coding region of GUS gene, the transposase of Ac element, the right border of Ds element and the promoter of Ubi gene were inserted into the T-DNA region of vector pCAMBIA1300 in turn to construct plasmid p13B. The orientations of the ubiquitons' promoter, Ac transposase and Bar are identical but opposite to that of the GUS gene (Fig.1). The plasmid p13B was then introduced into the calli of Oryza sativa subsp. japonica cv. Zhonghua 11 by Agrobacterium tumefaciens-mediated transforming to trap genes in rice. Eighteen independent transgenic lines were obtained and propagated. T(2) generations of 18 independent transgenic lines were screening by herbicide (Basta) (Fig.2) and the herbicide-resistant plants obtained were analyzed by PCR (Fig.3). Ds element transposed in an inheritable manner was found in 37 plants, in which 5 plants showed GUS activity (Fig.4).

The plasmid p13W8 carrying antisense fragment of waxy gene and plasmid pCAMBIA1300 containing hpt gene were introduced into rice by Agrobacterium tumefaciens-mediated co-transformation, and 86 transgenic plants were obtained, 32 of them showed positive bands for antisense waxy gene by PCR analysis, the waxy-positive plant frequency is 37.2%. The segregation of antisense fragment of waxy gene and hpt gene was observed by PCR using hpt gene primers and waxy gene primers respectively in 29 T(1) population. One hundred and eighty-three plants containing only the antisense fragment of waxy gene were identified in 1 264 T(1) plants, the waxy-positive plant frequency is 14.4% (Table 1). The amylose content of seeds derived from transgenic plants with only the antisense fragment of waxy gene were determined, varying degrees of reduction in amylose content were found in some plants (Table 2). Four T(1) plants with reduced amylose content were selected through anther culture. Thirty-four anther culture plants seed normally, 23 of them were shown to contain only the antisense fragment of waxy gene (Table 3) by PCR analysis, and the amylose content was reduced to 5%-12% (Table 4). It took only one and half years to obtain the stably inherited markerless transgenic rice with reduced amylose content by co-transformation and anther culture technique.

OsEBP-89 gene was an EREBP (ethylene responsive elements binding protein) transcription factor from rice (Oryza sativa). Northern blot analysis revealed that the expression of OsEBP-89 gene can be induced by ACC, 2,4-D, ABA, BR, JA, GA, 6-BA and salt. We found an ethylene responsive element-like ERE (ethylene responsive element), named IVC box, in the promoter region (-552 to -510) of OsEBP-89 gene. EMSA results showed the IVC box could specifically bind with nuclear proteins extracted from immature rice seeds. When the rice suspension cell harboring IVC/35S mini-promoter (-46)/GUS chimeric construct (pIVC/GUS) was treated by ACC, GUS activity could be induced. However, two plasmids with OsEBP-89 promoter/GUS (pPSG) and IVC box-deleted OsEBP-89 promoter/GUS (pSphSG) were transformed to rice, when both transgenic suspension cell treated with ACC the GUS expression was also inducible. This result indicated that there are more than one ethylene-responsive elements located in the promoter region of the OsEBP-89 gene.

It has been demonstrated in our previous work that rice OsBP-73 gene contains two exons interrupted by a 2471 bp intron. Here it was reported that the 5' flanking region of the ATG translation start codon in the first exon of OsBP-73 gene (from -1818 to +215) can not direct GUS gene expression in resistant rice calli or transgenic rice. When the complete OsBP-73 intron and its flanking region (from -1818 to +2844) are constructed in frame with GUS coding region, GUS activity can be detected in the resistant rice calli and transgenic rice. Experimental data also show that the complete OsBP-73 intron itself has no promoter activity. These results suggest that the OsBP-73 intron sequence is involved in directing the GUS gene expression. Another experiment demonstrates that the complete intron sequence can enhance the activity of OsEBP-89 gene promoter.