UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in carbohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recentl...UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in carbohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recently been reported to be essential for callose deposition during pollen mother cell and meiosis stages as well as for seed carbohydrate metabolism. In this study, a full-length cDNA of OsUgp2 was isolated from rice anther. Northern blot and RNA in situ hybridization indicated that the expression of OsUgp2 was preferentially in pollen and developmentally regulated. No transcripts were found in leaf, stem, lemma/palea, ripening grain and florets before the uninucleate microspore developmental stage, but a large quantity of OsUgp2 mRNA was found in pollen at the binucleate and mature stages. The immunolocalization of OsUgp2 showed a similar expression pattern to that by RNA in situ hybridization. The function of OsUgp2 was investigated by dsRNA-mediated transcriptional gene silencing. The pollen fertility of 16 independent transgenic rice plants was found between 25% and 90%, which was correlated with the amount of OsUgp2 mRNA. The results of morphological changes and starch variation during pollen development in transgenic rice showed that the abnormal feature of pollen development appeared after the uninucleate microspore stage. Starch failed to accumulate in pollen and thus led to sterile pollens. These results demonstrated that OsUgp2 is a pollen-preferential "late gene" and plays a key role during pollen maturation, especially for starch accumulation. OsUgp2 complements OsUgp1 to fulfill the UGPase’s functions necessary for the full process of pollen development.展开更多
Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice （Oryza sativa L. ssp. japonica）. The antisense Wx...Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice （Oryza sativa L. ssp. japonica）. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels ofamylose content were similar to the control in 19 lines （16.0%）; 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was conc展开更多
Rice (Oryza sativa L.) eating and cooking quality is mainly influenced by its starch properties. Mapping quantitative trait loci (QTL) for starch properties not only helps us understand their genetic basis leading to ...Rice (Oryza sativa L.) eating and cooking quality is mainly influenced by its starch properties. Mapping quantitative trait loci (QTL) for starch properties not only helps us understand their genetic basis leading to acceleration of quality improvement, but also helps us find possible genes participating in the synthesis of starch. A recombinant inbred line (RIL) population consisting of 107 lines, derived from an indica (Zaiyeqing 8, ZYQ 8) and a japonica (Jingxi 17, JX 17) rice, was used to investigate the genetic factors affecting starch quality parameters, such as apparent amylose content (AAC), gel consistency (GC), starch pasting viscosity parameters, gel textural properties, gelatinization temperature (GT) and starch retrogradation properties. A total of 44 QTLs covered chromosomes 2-6, 8, 9 and 11 were detected for the 22 traits, with at least one QTL and as many as four QTLs for each individual trait. The results indicated that two major genes were responsible for most starch property traits. The Wx gene that encodes granule bound starch synthase on chromosome 6 was significant for AAC, GC, starch pasting viscosity parameters, gel textural properties and starch retrogradation properties. The alk gene linked with Wx on chromosome 6 was significant for starch gelatinization temperature characteristics. All other QTLs were minor genes. One QTL on chromosome 9 flanked by RZ404 and G295 was significant for gel hardness (HD), gumminess (GUM), chewiness (CHEW), peak temperature of retrogradated starch (RTp), and percentage retrogradation (R%) and all these traits were not tested before.展开更多
The waxy gene(Wx) in rice, which encodes the granule bound starch synthase enzyme, is responsible for amylose synthesis. Glutinous(sticky) rice has little or no amylose that can be used in various applications, such a...The waxy gene(Wx) in rice, which encodes the granule bound starch synthase enzyme, is responsible for amylose synthesis. Glutinous(sticky) rice has little or no amylose that can be used in various applications, such as brewing. In this study, knockout of the Wx gene with CRISPR/Cas9 technology was conducted in two elite japonica rice lines, Huaidao 5(HD5) and Suken 118(SK118), aiming to develop elite sticky rice varieties. We achieved six homozygous T_0 plants with more than 200 bp deletion in the Wx gene, as well as 36 wx-HD5 and 18 wx-SK118 homozygous transgene-free plants in the T_1 generation. The seeds of all the mutants were white and opaque, similar to those of sticky rice, and contained only 2.6%–3.2% amylose. Results of scanning electron microscopy showed that the quality of rice did not change. In conclusion, we successfully developed two elite sticky rice varieties.展开更多
Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were f...Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.展开更多
A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these gen...A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.展开更多
Amylose content(AC)is a crucial determinant of the eating and cooking quality(ECQ)of rice,with low AC varieties exhibiting a softer texture and greater stickiness-attributes that enhance palatability and are desirable...Amylose content(AC)is a crucial determinant of the eating and cooking quality(ECQ)of rice,with low AC varieties exhibiting a softer texture and greater stickiness-attributes that enhance palatability and are desirable in specific culinary contexts.To harness these traits,significant efforts have been made to manipulate AC to improve rice ECQ.Our research utilized the MutMap+approach to identify LAC6/TL1,a gene that is an allele of Du13,responsible for low AC.LAC6 encodes a C2H2 zinc finger protein,which specifically increases the splicing efficiency of the Wxb allele without affecting the Wxa allele.Functional studies of LAC6 revealed that its proper integration could rectify the undesirable AC phenotype,whereas mutations within this gene led to reduced AC and were associated with shorter grain length and decreased thousand-grain weight.Despite these drawbacks,such mutations positively impact rice palatability,presenting a trade-off between grain size and eating quality.To address the challenges posed by the reduced grain weight associated with LAC6 mutations,we developed a specific molecular marker for LAC6,which has been effectively used in breeding programs to select lac6/tl1/du13 homozygous individuals with larger grain size.Our findings demonstrate that the“small grain”trait associated with lac6/tl1/du13 can be effectively mitigated through combined phenotype-based and marker-assisted selection.This study highlights the potential of lac6/tl1/du13 as a valuable gene for breeding novel,high-quality soft rice varieties through targeted breeding strategies.展开更多
Rice Wx genes have been cloned and sequenced from Oryza saliva subsp. japonica (Wx-R-jp) and subsp. indica (Wx-R-id) and wild rice Oryza saliva L. f. spontanea (Wx-R-sp). Comparison of the nucleotide sequences of thes...Rice Wx genes have been cloned and sequenced from Oryza saliva subsp. japonica (Wx-R-jp) and subsp. indica (Wx-R-id) and wild rice Oryza saliva L. f. spontanea (Wx-R-sp). Comparison of the nucleotide sequences of these three Wx genes indicates that Wx-R-jp and Wx-R-id genes contain two types of transposon-like elements, designated by RTL-1 and RTL-2. in the region of their two introns and 5’ upstream, RTL-1 shares sequence homology with valine tRNA molecules, and contains an internal promoter of RNA polymerase Ⅲ. RTL-2 forms a stem-loop structure. Both RTL-1 and RTL-2 are flanked by direct repetitive sequences. Compared with the elements that have been known transposable, RTL-1 resembles the short interspersed-repeated DNA elements (SINEs) and RTL-2 is similar to the fold-back (FB) element of trahsposon of Drosophila.展开更多
The complete nucleotide (nt) sequence of the rice waxy(Wx) gene, which is responsible for the synthesis of amylose in endosperm and pollen, has been determined by a combination of restriction mapping and nt sequence a...The complete nucleotide (nt) sequence of the rice waxy(Wx) gene, which is responsible for the synthesis of amylose in endosperm and pollen, has been determined by a combination of restriction mapping and nt sequence analysis of two overlapping genomic DNA clones. The entire gene is about 5.5 kb in length. The alignment of the nt sequence of the Wx gene from rice with those of maize (Klsgen, R. B. et al.) and barley (Rohde, W. et al.) revealed the presence of thirteen introns and fourteen exons. The full-length of Wx protein in cluding transit peptide is 609 amino acid (aa) residues. The calculated molecular weight of rice Wx preprotein is about 72 kD. There is no significant difference between the similarity scores of the aa sequence deduced from the rice Wx gene compared with those of maize and barley. However, the nt sequences of the 5'-end upstream, 3'-end downstream and introns of the rice Wx gene, as well as the aa sequence of the transit peptide region of the Wx preprotein have low similarity scores in comparison with those of maize and barley.展开更多
基金Supported by National Natural Science Foundation of China (Grant No. 30370800)Changjiang Scholars and Innovative Research Team from Ministry of Education in China (Grant No. IRT0448)
文摘UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in carbohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recently been reported to be essential for callose deposition during pollen mother cell and meiosis stages as well as for seed carbohydrate metabolism. In this study, a full-length cDNA of OsUgp2 was isolated from rice anther. Northern blot and RNA in situ hybridization indicated that the expression of OsUgp2 was preferentially in pollen and developmentally regulated. No transcripts were found in leaf, stem, lemma/palea, ripening grain and florets before the uninucleate microspore developmental stage, but a large quantity of OsUgp2 mRNA was found in pollen at the binucleate and mature stages. The immunolocalization of OsUgp2 showed a similar expression pattern to that by RNA in situ hybridization. The function of OsUgp2 was investigated by dsRNA-mediated transcriptional gene silencing. The pollen fertility of 16 independent transgenic rice plants was found between 25% and 90%, which was correlated with the amount of OsUgp2 mRNA. The results of morphological changes and starch variation during pollen development in transgenic rice showed that the abnormal feature of pollen development appeared after the uninucleate microspore stage. Starch failed to accumulate in pollen and thus led to sterile pollens. These results demonstrated that OsUgp2 is a pollen-preferential "late gene" and plays a key role during pollen maturation, especially for starch accumulation. OsUgp2 complements OsUgp1 to fulfill the UGPase’s functions necessary for the full process of pollen development.
基金supported by the grants from the Ministry of Science and Technology,China(ZJY-A-02-02)the Shanghai Science Committee Program Fund,China(013912019)Natural Science Fundation of Jiangsu Province,China(BK2007510)
文摘Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice （Oryza sativa L. ssp. japonica）. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels ofamylose content were similar to the control in 19 lines （16.0%）; 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was conc
文摘Rice (Oryza sativa L.) eating and cooking quality is mainly influenced by its starch properties. Mapping quantitative trait loci (QTL) for starch properties not only helps us understand their genetic basis leading to acceleration of quality improvement, but also helps us find possible genes participating in the synthesis of starch. A recombinant inbred line (RIL) population consisting of 107 lines, derived from an indica (Zaiyeqing 8, ZYQ 8) and a japonica (Jingxi 17, JX 17) rice, was used to investigate the genetic factors affecting starch quality parameters, such as apparent amylose content (AAC), gel consistency (GC), starch pasting viscosity parameters, gel textural properties, gelatinization temperature (GT) and starch retrogradation properties. A total of 44 QTLs covered chromosomes 2-6, 8, 9 and 11 were detected for the 22 traits, with at least one QTL and as many as four QTLs for each individual trait. The results indicated that two major genes were responsible for most starch property traits. The Wx gene that encodes granule bound starch synthase on chromosome 6 was significant for AAC, GC, starch pasting viscosity parameters, gel textural properties and starch retrogradation properties. The alk gene linked with Wx on chromosome 6 was significant for starch gelatinization temperature characteristics. All other QTLs were minor genes. One QTL on chromosome 9 flanked by RZ404 and G295 was significant for gel hardness (HD), gumminess (GUM), chewiness (CHEW), peak temperature of retrogradated starch (RTp), and percentage retrogradation (R%) and all these traits were not tested before.
基金supported by the National Key Research and Development Program (Grant No. 2017YFD0100403)the Jiangsu Province Key Research and Development Program (Modern Agriculture) Project (Grant No. BE2017345-2)+1 种基金the Exploratory Project of the Jiangsu Academy of Agricultural Sciences [Grant No. ZX(17)2014]the Jiangsu Province Natural Science Foundation (Grant No. BK20171326)
文摘The waxy gene(Wx) in rice, which encodes the granule bound starch synthase enzyme, is responsible for amylose synthesis. Glutinous(sticky) rice has little or no amylose that can be used in various applications, such as brewing. In this study, knockout of the Wx gene with CRISPR/Cas9 technology was conducted in two elite japonica rice lines, Huaidao 5(HD5) and Suken 118(SK118), aiming to develop elite sticky rice varieties. We achieved six homozygous T_0 plants with more than 200 bp deletion in the Wx gene, as well as 36 wx-HD5 and 18 wx-SK118 homozygous transgene-free plants in the T_1 generation. The seeds of all the mutants were white and opaque, similar to those of sticky rice, and contained only 2.6%–3.2% amylose. Results of scanning electron microscopy showed that the quality of rice did not change. In conclusion, we successfully developed two elite sticky rice varieties.
基金supported by funding from the National Natural Science Foundation of China(31801016)the Agricultural Variety Improvement Project of Shandong Province(2019LZGC015)。
文摘Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39893320).
文摘A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.
基金supported by the National Key Research and Development Program of China(2023YFD1200901)the National Natural Science Foundation of China(32230074 and 32161143004)+3 种基金the Research Programs from Jiangsu Government(JBGS[2021]001,BZ2021017,and Qing Lan Project)the Foreign Expert Project(G2023014064L)Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX21_3239)the Yangzhou University High-end Talent Program.
文摘Amylose content(AC)is a crucial determinant of the eating and cooking quality(ECQ)of rice,with low AC varieties exhibiting a softer texture and greater stickiness-attributes that enhance palatability and are desirable in specific culinary contexts.To harness these traits,significant efforts have been made to manipulate AC to improve rice ECQ.Our research utilized the MutMap+approach to identify LAC6/TL1,a gene that is an allele of Du13,responsible for low AC.LAC6 encodes a C2H2 zinc finger protein,which specifically increases the splicing efficiency of the Wxb allele without affecting the Wxa allele.Functional studies of LAC6 revealed that its proper integration could rectify the undesirable AC phenotype,whereas mutations within this gene led to reduced AC and were associated with shorter grain length and decreased thousand-grain weight.Despite these drawbacks,such mutations positively impact rice palatability,presenting a trade-off between grain size and eating quality.To address the challenges posed by the reduced grain weight associated with LAC6 mutations,we developed a specific molecular marker for LAC6,which has been effectively used in breeding programs to select lac6/tl1/du13 homozygous individuals with larger grain size.Our findings demonstrate that the“small grain”trait associated with lac6/tl1/du13 can be effectively mitigated through combined phenotype-based and marker-assisted selection.This study highlights the potential of lac6/tl1/du13 as a valuable gene for breeding novel,high-quality soft rice varieties through targeted breeding strategies.
基金Project supported by grants for the High Technology Development Programme of China and the Rockefeller Foundation.
文摘Rice Wx genes have been cloned and sequenced from Oryza saliva subsp. japonica (Wx-R-jp) and subsp. indica (Wx-R-id) and wild rice Oryza saliva L. f. spontanea (Wx-R-sp). Comparison of the nucleotide sequences of these three Wx genes indicates that Wx-R-jp and Wx-R-id genes contain two types of transposon-like elements, designated by RTL-1 and RTL-2. in the region of their two introns and 5’ upstream, RTL-1 shares sequence homology with valine tRNA molecules, and contains an internal promoter of RNA polymerase Ⅲ. RTL-2 forms a stem-loop structure. Both RTL-1 and RTL-2 are flanked by direct repetitive sequences. Compared with the elements that have been known transposable, RTL-1 resembles the short interspersed-repeated DNA elements (SINEs) and RTL-2 is similar to the fold-back (FB) element of trahsposon of Drosophila.
基金Project supported by the grants from the High Technology Development Programme of China and the Rockefeller Foundation.
文摘The complete nucleotide (nt) sequence of the rice waxy(Wx) gene, which is responsible for the synthesis of amylose in endosperm and pollen, has been determined by a combination of restriction mapping and nt sequence analysis of two overlapping genomic DNA clones. The entire gene is about 5.5 kb in length. The alignment of the nt sequence of the Wx gene from rice with those of maize (Klsgen, R. B. et al.) and barley (Rohde, W. et al.) revealed the presence of thirteen introns and fourteen exons. The full-length of Wx protein in cluding transit peptide is 609 amino acid (aa) residues. The calculated molecular weight of rice Wx preprotein is about 72 kD. There is no significant difference between the similarity scores of the aa sequence deduced from the rice Wx gene compared with those of maize and barley. However, the nt sequences of the 5'-end upstream, 3'-end downstream and introns of the rice Wx gene, as well as the aa sequence of the transit peptide region of the Wx preprotein have low similarity scores in comparison with those of maize and barley.
