Comparison of Waxy gene regulation in the endosperm and pollen in Oryza sativa L

The Waxy (Wx) gene controls amylose synthesis in rice (Oryza sativa) and its expression is regulated organ-specifically. The Wx gene is expressed in the endosperm and pollen but not in other organs. In order to know whether Wx gene regulation is the same in the endosperm and pollen, we compared expression patterns of the rice Wx gene in these two organs by immunoblot analysis. We focused on the allelic differences (Wxa and Wxb), cool temperature response and effects of the mutation at the du loci. The results obtained are as follows. First, the quantitative regulation depending on two alleles, Wxa and Wxb, was common to both organs; Wx protein levels from the Wxa allele were about 10-fold higher than those from the Wxb allele in the pollen as well as in the endosperm. Second, in both the endosperm and pollen, expression of the Wxb gene, but not the Wxa gene, was enhanced in response to cool temperature. In contrast to these two types of regulation, analysis of two du mutants, 2035 (du1) and 76-3 (du2), revealed that the pattern of reduction in Wx protein levels in the pollen was distinct from that in the endosperm, suggesting that functions of the two du+ genes differ in these two organs.     

Introduction of Wx transgene into rice wx mutants leads to both high- and low-amylose rice

The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. Two functional Wx alleles of rice exist: Wx(a), which produces a large amount of amylose, and Wx(b), which produces a smaller amount of amylose because of the mutation at the 5' splice site of intron 1. Wx(b) is largely distributed in Japonica cultivars, and high amylose cultivars do not exist in Japonica cultivars. We introduced the cloned Wx(a) cDNA into null-mutant Japonica rice (wx). The amylose contents of these transgenic plants were 6-11% higher than that of the original cultivar, Labelle, which carries the Wx(a) allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle. We also observed a gene-dosage effect of the Wx(a) transgene on Wx protein expression, but a smaller dosage effect was observed in amylose production with over 40% of amylose content in transgenic rice. Moreover, one transgenic line carrying eleven copies of the transgene showed low levels of Wx expression and amylose in the endosperm. This suggested that the integration of excessive copies of the transgene might lead to gene silencing.     

Subspecies-specific intron length polymorphism markers reveal clear genetic differentiation in common wild rice （Oryza rufipogon L.） in relation to the domestication of cultivated rice （O. sativa L.）

It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice （O. sativa）. However, how the two subspecies of O. sativa （indica and japonica） were domesticated has long been debated. To investigate the genetic differentiation in O. rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism （SSILP） markers by comparison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that the indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa. Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups： one group （Group I） comprises all the O. rufipogon accesions from tropical （South and Southeast） Asia as well as the standard indica rice; the other group （Group II） comprises all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have significantly higher frequencies of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domestication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. ruffpogon might occur after its speciation from other AA-genome species.     

Genealogy of the "Green Revolution" gene in rice

During the "Green Revolution" of rice, high-yielding varieties (HYVs) were developed using a semi-dwarf gene (sd1 or OsGA20ox2). The presence or absence of the two mutant alleles (DGWG type in Dee-geo-woo-gen and JKK type in Jikkoku) were surveyed by PCR using 256 accessions of eight wild and two cultivate rice species. The DGWG allele was detected in a landrace (Oryza sativa) and two accessions of wild rice (O. rufipogon), all of which are from China, showing their limited distribution. Genealogical studies of the OsGA20ox2 gene showed that the 62 sequences of O. sativa and O. rufipogon included 20 distinct haplotypes, indicating that the species complex contained OsGA20ox2 genes from two different lineages. The silent site nucleotide diversities (pi and theta(w)) were extremely low in Japonica rice, suggesting a genetic bottleneck. The haplotype network showed that the DGWG and JKK alleles were derived in different lineages. The DGWG carrier (W1944) had unique polymorphisms in the surrounding region of the locus, suggesting that the DGWG allele has been preserved in the wild progenitor, rather than that the DGWG allele has been introgressed from HYVs to W1944. Although a semi-dwarfing plant is a weak competitor under saturated fields, the crossing experiment revealed that the DGWG variant might have been preserved as a hidden variation in the genetic background of wild rice, without expressing a short-stature.     

云南元江普通野生稻群体Wx基因（CT）n重复序列和第一内含子G/T位碱基分析

对云南元江普通野生稻(O.rufipogon)90个个体Wx基因区段内的重复序列（CT）n和第一内含子供体+1位碱基G/T分别进行了比较分析。结果表明云南元江普通野生稻三个居群中的90个单株在重复序列（CT）n和G/T位点纯合一致,没有多态性;其G/T位点碱基均为G;其重复序列（CT）n基因型与云南地方籼稻品种优势基因型相似但又有所区别。本研究结果为云南元江普通野生稻Wx基因利用和在稻种进化上的地位提供了信息。     

籼稻蜡质基因5＇上游区与GUS基因编码区融合基因的构建和在水稻中的瞬间表达

为了鉴定水稻蜡质基因５＇上游区的顺式作用因子与研究它们在组织专一性表达中的作用,我们对籼稻品种２３２的蜡质基因５＇上游－１１５至－２１２０的区域进行了顺序测定,并将此基因的５＇上游区同报告基因ＧＵＳ构建成融合基因,用基因枪粒子轰击的方法将此融合基因导入水稻未成熟种子的幼胚和糊粉层细胞中,瞬间表达检测的结果表明,我们构建的融合基因中蜡质基因５＇上游区的长度已足以使ＧＵＳ基因在上述组织中表达。     

Polyphyletic origin of cultivated rice: based on the interspersion pattern of SINEs

The wild rice species Oryza rufipogon with wide intraspecific variation is thought to be the progenitor of the cultivated rice species Oryza sativa with two ecotypes, japonica and indica. To determine the origin of cultivated rice, subfamily members of the rice retroposon p-SINE1, which show insertion polymorphism in the O. sativa -O. rufipogon population, were identified and used to "bar code" each of 101 cultivated and wild rice strains based on the presence or absence of the p-SINE1 members at the respective loci. A phylogenetic tree constructed based on the bar codes given to the rice strains showed that O. sativa strains were classified into two groups corresponding to japonica and indica, whereas O. rufipogon strains were in four groups, in which annual O. rufipogon strains formed a single group, differing from the perennial O. rufipogon strains of the other three groups. Japonica strains were closely related to the O. rufipogon perennial strains of one group, and the indica strains were closely related to the O. rufipogon annual strains, indicating that O. sativa has been derived polyphyletically from O. rufipogon. The subfamily members of p-SINE1 constitute a powerful tool for studying the classification and relationship of rice strains, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains.     

云南元江普通野生稻群体Wx基因(CT)_n重复序列和第1内含子G/T位碱基分析

对云南元江普通野生稻90个个体Wx基因区段内的重复序列(CT)n和第1内含子供体+1位碱基G/T分别进行了比较分析。结果表明云南元江普通野生稻3个居群中的90个单株在重复序列(CT)n和G/T位点纯合一致,没有多态性;其G/T位点碱基均为G;其重复序列(CT)n基因型与云南地方籼稻品种优势基因型相似但又有所区别。本研究结果为云南元江普通野生稻Wx基因利用和在稻种进化上的地位提供了信息。     

A rice blast-resistance genetic resource from wild rice in Yunnan, China.

Compared to Pi-ta(-) alleles, Pi-ta(+) alleles can cause blast resistance response. In this work, Pi-ta gene in multiple rice materials, including local rice cultivars, different types of O. rufipogon and O. longistaminata was detected by molecular cloning and sequence analysis. Results indicated that Pi-ta(+) alleles were rare alleles, because in all the tested materials, only the 'Erect' type of O. rufipogon (ETOR) from Jinghong county in Yunnan province contains a Pi-ta(+) allele. Another rice blast resistance gene, Pib, confers resistance to the Japanese strain of M. grisea, was also confirmed to be functional in this type of O. rufipogon. The results of pathogen inoculation test show that ETOR is more strongly resistant to the tested blast pathogen races than other types of O. rufipogon. The resistance of ETOR may at least partially depend upon the functioning of Pi-ta and Pib gene. As O. rufipogon has the same type of genome with the cultivated rice (O. sativa), Pi-ta(+) and Pib gene in Erect type of O. rufipogon can be used to improve the tolerance of cultivated rice to blast, either by traditional hybridization or by genetic engineering.     

The serine/arginine-rich protein family in rice plays important roles in constitutive and alternative splicing of pre-mRNA

Ser/Arg-rich (SR) proteins play important roles in the constitutive and alternative splicing of pre-mRNA. We isolated 20 rice (Oryza sativa) genes encoding SR proteins, of which six contain plant-specific characteristics. To determine whether SR proteins modulate splicing efficiency and alternative splicing of pre-mRNA in rice, we used transient assays in rice protoplasts by cotransformation of SR protein genes with the rice Waxy(b) (Wx(b))-beta-glucuronidase fusion gene. The results showed that plant-specific RSp29 and RSZp23, an SR protein homologous to human 9G8, enhanced splicing and altered the alternative 5' splice sites of Wx(b) intron 1. The resulting splicing pattern was unique to each SR protein; RSp29 stimulated splicing at the distal site, and RSZp23 enhanced splicing at the proximal site. Results of domain-swapping experiments between plant-specific RSp29 and SCL26, which is a homolog of human SC35, showed the importance of RNA recognition motif 1 and the Arg/Ser-rich (RS) domain for the enhancement of splicing efficiencies. Overexpression of plant-specific RSZ36 and SRp33b, a homolog of human ASF/SF2, in transgenic rice changed the alternative splicing patterns of their own pre-mRNAs and those of other SR proteins. These results show that SR proteins play important roles in constitutive and alternative splicing of rice pre-mRNA.     

RAPD, RFLP and SSLP analyses of phylogenetic relationships between cultivated and wild species of rice.

RAPD, RFLP, nuclear SSLP and chloroplast SSLP analyses were carried out to clarify the phylogenetic relationships among A-genome species of rice. In total, 12 cultivars of Oryza sativa (4 Japonica, 3 Javanica and 5 Indica), one cultivar of O. glaberrima, and 17 wild accessions (12 O. rufipogon, 2 O. glumaepatula, 1 O. longistaminata, 1 O. meridionalis and 1 O. barthii) were used. Their banding patterns were scored and compared to evaluate the similarity between accessions. Genetic differentiation within and between taxa was examined based on the average similarity indices. Except for chloroplast SSLP analysis, the average similarities were higher within O. sativa than within O. rufipogon, and O. sativa Indica had greater intrasubspecific variation than Japonica and Javanica. Comparisons between cultivated and wild species showed that O. sativa was closely related to O. rufipogon, while O. glaberrima was closely related to O. barthii. This indicated that two cultivated species, O. sativa and O. glaberrima, originated from O. rufipogon and O. barthii, respectively. Domestication of O. sativa seemed to be diphyletic, since strong similarity was observed between O. sativa Japonica-Javanica and O. rufipogon from China and between O. sativa Indica and O. rufipogon from tropical Asia. In addition, dendrograms for RAPD, RFLP, and nuclear and chloroplast SSLP analyses were constructed to reveal the overall genetic relationships among A-genome species. In all analyses, O. sativa and O. glaberrima formed groups with O. rufipogon and O. barthii, respectively. However, their manners of clustering with other wild species were not the same. The results of RAPD and RFLP analyses indicate that O. glumaepatula was relatively close to the groups of O. sativa and O. glaberrima whereas O. longistaminata and O. meridionalis were highly differentiated from other A-genome species. On the other hand, clear interspecific relationships were not obtained by nuclear or chloroplast SSLP analyses.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.