Isoamylase 1(ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associat...Isoamylase 1(ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated endonuclease 9(CRISPR/Cas9) system was used to edit ISA1 gene in rice via Agrobacterium-mediated transformation. We identified 36 genetic edited lines from 55 T_0 transgenic events, and classified the mutation forms into 7 types. Of those, two homozygous mutants, cr-isa1-1(type 1, with an adenine insertion) and cr-isa1-2(type 3, with a cytosine deletion) were selected for further analysis. Seed sizes of both cr-isa1-1 and cr-isa1-2 were affected, and the two mutants also displayed a shrunken endosperm with significantly lower grain weight. Electron microscopy analysis showed that abnormal starch granules and amyloplasts were found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants were significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild-type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants were also altered. Moreover, transcript levels of most starch synthesis-related genes were significantly lower in cr-isa1 mutants. In conclusion, the results indicated that gene edition of ISA1 affected starch synthesis and endosperm development, and brought potential implications for rice quality breeding.展开更多
We here reported the identification and characterization of a novel gain-of-function spotted-leaf 26 (Spl26) mutant from an ethylmethylsulfone (EMS)-induced rice cultivar IR64. Spl26 displayed reddish- brown lesions t...We here reported the identification and characterization of a novel gain-of-function spotted-leaf 26 (Spl26) mutant from an ethylmethylsulfone (EMS)-induced rice cultivar IR64. Spl26 displayed reddish- brown lesions that firstly appeared on the leaf tips at the early tillering stage and spread gradually downward to cover the whole leaf blades that wilted subsequently. The lesion development was light- dependent under natural conditions. Spl26 exhibited impaired photosynthetic capacity with decreased chlorophyll content and lowered photosynthetic parameters which ultimately led to the poor performance of agronomic traits. Severe cell death occurred in Spl26 in accompany with increased malonaldehyde level and membrane ion leakage rate, elevated reactive oxygen species (ROS) accumulation, altered ROS scavenging activities, increased DNA fragmentation and decreased soluble protein levels. Defense responses were activated in Spl26 with enhanced resistance to rice bacterial blight, up-regulation of defense response genes and altered endogenous hormone levels. The spotted-leaf phenotype is controlled by a single dominant nuclear gene localized to a 305 kb region between RM5490 and InDel42 on the short arm of chromosome 7. The data suggested that Spl26 is a novel gain-of-function spotted-leaf mutant with enhanced bacterial disease resistance and immunity-associated premature leaf senescence and would provide the basis for cloning of the target gene.展开更多
Rice grain chalkiness is an important characteristic,but the difference between chalky and translucent parts in grains is still unclear.Here,we investigated the differences of flour made from the chalky or translucent...Rice grain chalkiness is an important characteristic,but the difference between chalky and translucent parts in grains is still unclear.Here,we investigated the differences of flour made from the chalky or translucent part of rice grains in three indica and three japonica rice varieties.The chalky flour had significantly lower amylose and protein contents and looser starch granule morphology,and starches in the chalky flour had higher relative crystallinity,higher short chain content but lower long chain content than those in the translucent flour.The water states,determined with nuclear magnetic resonance,differed between the chalky and translucent flour after soaking,cooking and retrograding,and the chalky flour had more bound and free water but less constructural water than the translucent flour.Mostly,the chalky flour had lower viscosity and shorter gel consistency,but higher onset temperature and gelatinization enthalpy than the translucent flour.The results indicated that starch granule morphology would be more indicative than other attributions on pasting and gelatinization properties of chalky and translucent parts of rice grains.展开更多
OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a cha...OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a chalky endosperm with a white core,which significantly decreased 1000-grain weight.In addition,many rounded starch granules and abnormal amyloplasts were present in the central region of mutant endosperm cells with increased amylose and lipid contents,decreased total protein content,and altered physicochemical properties of starch.The OsPho1 protein is localized in chloroplasts,and quantitative real-time PCR(qRT-PCR)andβ-glucuronidase(GUS)staining indicated that OsPho1 was highly expressed in seeds at 5 d after fertilization(DAF).OsPho1 mutations displayed close relationships with plastidial phosphoglucomutase and ADPGlc pyrophosphorylase based onα-D-glucose-1P at different temperatures.Moreover,the expressions of starch metabolismrelated genes were also altered in the mutant,and the overexpression of OsPho1 may cause grain chalkiness.展开更多
Amylose content(AC)is the key determinant of eating and cooking quality(ECQ)of rice.The major Wxa allele of granule-bound starch synthase I(GBSS I)in indica rice produces higher AC,making rice hard and dry after cooki...Amylose content(AC)is the key determinant of eating and cooking quality(ECQ)of rice.The major Wxa allele of granule-bound starch synthase I(GBSS I)in indica rice produces higher AC,making rice hard and dry after cooking.Recent work has improved ECQ of japonica rice via clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)or cytosine base editing(CBE)techniques.展开更多
基金supported by the National Nature Science Foundation of China (Grant Nos. 31471472 and 31521064)the National S&T Major Project of China (Grant No. 2016ZX08001006)the Central Level, Non-Profit, Scientific Research Institutes Basic R and D Operations Special Fund (Grant Nos.Y2017PT46 and 2017RG002-1)
文摘Isoamylase 1(ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated endonuclease 9(CRISPR/Cas9) system was used to edit ISA1 gene in rice via Agrobacterium-mediated transformation. We identified 36 genetic edited lines from 55 T_0 transgenic events, and classified the mutation forms into 7 types. Of those, two homozygous mutants, cr-isa1-1(type 1, with an adenine insertion) and cr-isa1-2(type 3, with a cytosine deletion) were selected for further analysis. Seed sizes of both cr-isa1-1 and cr-isa1-2 were affected, and the two mutants also displayed a shrunken endosperm with significantly lower grain weight. Electron microscopy analysis showed that abnormal starch granules and amyloplasts were found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants were significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild-type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants were also altered. Moreover, transcript levels of most starch synthesis-related genes were significantly lower in cr-isa1 mutants. In conclusion, the results indicated that gene edition of ISA1 affected starch synthesis and endosperm development, and brought potential implications for rice quality breeding.
基金supported by the Ministry of Science and Technology of China(Grant No.2016YFD0101104)
文摘We here reported the identification and characterization of a novel gain-of-function spotted-leaf 26 (Spl26) mutant from an ethylmethylsulfone (EMS)-induced rice cultivar IR64. Spl26 displayed reddish- brown lesions that firstly appeared on the leaf tips at the early tillering stage and spread gradually downward to cover the whole leaf blades that wilted subsequently. The lesion development was light- dependent under natural conditions. Spl26 exhibited impaired photosynthetic capacity with decreased chlorophyll content and lowered photosynthetic parameters which ultimately led to the poor performance of agronomic traits. Severe cell death occurred in Spl26 in accompany with increased malonaldehyde level and membrane ion leakage rate, elevated reactive oxygen species (ROS) accumulation, altered ROS scavenging activities, increased DNA fragmentation and decreased soluble protein levels. Defense responses were activated in Spl26 with enhanced resistance to rice bacterial blight, up-regulation of defense response genes and altered endogenous hormone levels. The spotted-leaf phenotype is controlled by a single dominant nuclear gene localized to a 305 kb region between RM5490 and InDel42 on the short arm of chromosome 7. The data suggested that Spl26 is a novel gain-of-function spotted-leaf mutant with enhanced bacterial disease resistance and immunity-associated premature leaf senescence and would provide the basis for cloning of the target gene.
基金supported by the National Natural Science Foundation of China (Grant Nos. 32172080 and 31971925)China Agriculture Research System (Grant No. CARS-01-16)
文摘Rice grain chalkiness is an important characteristic,but the difference between chalky and translucent parts in grains is still unclear.Here,we investigated the differences of flour made from the chalky or translucent part of rice grains in three indica and three japonica rice varieties.The chalky flour had significantly lower amylose and protein contents and looser starch granule morphology,and starches in the chalky flour had higher relative crystallinity,higher short chain content but lower long chain content than those in the translucent flour.The water states,determined with nuclear magnetic resonance,differed between the chalky and translucent flour after soaking,cooking and retrograding,and the chalky flour had more bound and free water but less constructural water than the translucent flour.Mostly,the chalky flour had lower viscosity and shorter gel consistency,but higher onset temperature and gelatinization enthalpy than the translucent flour.The results indicated that starch granule morphology would be more indicative than other attributions on pasting and gelatinization properties of chalky and translucent parts of rice grains.
基金supported by the National Science Foundation of China(Grant No.3197150429).
文摘OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a chalky endosperm with a white core,which significantly decreased 1000-grain weight.In addition,many rounded starch granules and abnormal amyloplasts were present in the central region of mutant endosperm cells with increased amylose and lipid contents,decreased total protein content,and altered physicochemical properties of starch.The OsPho1 protein is localized in chloroplasts,and quantitative real-time PCR(qRT-PCR)andβ-glucuronidase(GUS)staining indicated that OsPho1 was highly expressed in seeds at 5 d after fertilization(DAF).OsPho1 mutations displayed close relationships with plastidial phosphoglucomutase and ADPGlc pyrophosphorylase based onα-D-glucose-1P at different temperatures.Moreover,the expressions of starch metabolismrelated genes were also altered in the mutant,and the overexpression of OsPho1 may cause grain chalkiness.
基金supported by the China National Key Research and Development Program(Grant No.2020YFE0202300)the Central Public-Interest Scientific Institution Basal Research Fund of China(Grant No&Y2020PT07 and Y2020YJ09)the International Science&Technology Innovation Program of Chinese Academy of Agricultural Sciences,China(Grant No.CAAS-ZDRW202109).
文摘Amylose content(AC)is the key determinant of eating and cooking quality(ECQ)of rice.The major Wxa allele of granule-bound starch synthase I(GBSS I)in indica rice produces higher AC,making rice hard and dry after cooking.Recent work has improved ECQ of japonica rice via clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)or cytosine base editing(CBE)techniques.
