Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Lipoxygenase (LOX, EC1.13.11.12) is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a(+) and transformed into Escherichia coli BL21 (DE3). Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside (IPTG) and the purified recombinant protein was obtained by His·Bind? Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 (acetate buffer) and the optimum temperature was 30°C for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

OsHemA gene, encoding glutamyl-tRNA reductase(GluTR) is essential for chlorophyll biosynthesis in rice(Oryza sativa)

Chlorophyll(Chl) biosynthesis is essential for photosynthesis and plant growth.Glutamyl-tRNA reductase(GluTR) catalyzes glutamyl-tRNA into glutamate-1-semialdehyde(GSA) and initiates the chlorophyll biosynthesis.Even though the main role of GluTR has been established,the effects caused by natural variations in its corresponding gene remain largely unknown.Here,we characterized a spontaneous mutant in paddy field with Chl biosynthesis deficiency,designated as cbd1.With intact thylakoid lamellar structure,the cbd1 plant showed light green leaves and reduced Chl and carotenoids(Cars) content significantly compared to the wild type.By map-based gene cloning,the mutation was restricted within a 57-kb region on chromosome 10,in which an mPingA miniature inverted-repeat transposable element(MITE) inserted in the promoter region of OsHemA gene.Both leaf color and the pigment contents in cbd1 were recovered in a complementation test,confirming OsHemA was responsible for the mutant phenotype.OsHemA was uniquely predicted to encode GluTR and its expression level was dramatically repressed in cbd1.Transient transformation in protoplasts demonstrated that GluTR localized in chloroplasts and a signal peptide exists in its N-terminus.A majority of Chl biosynthesis genes,except for POR and CHLG,were down-regulated synchronously by the repression of OsHemA,suggesting that an attenuation occurred in the Chl biosynthesis pathway.Interestingly,we found major agronomic traits involved in rice yield were statistically unaffected,except for the number of full grains per panicle was increased in cbd1.Collectively,OsHemA plays an essential role in Chl biosynthesis in rice and its weak allele can adjust leaf color and Chls content without compromise to rice yield.

Progress and Prospect of Functional Rice Researches

With the rapid development of economy and the increase of people's living standards,functional foods are being gradually embraced by consumers and have a great growth potential. Rice is the main staple food in China. It plays an important role in promoting people's health and in improving public nutrition,and therefore there is an ever-increasing consumer demand for rice for its functional quality. This study introduces the concept of functional rice and summarizes the current research progress on this topic and the application of breeding strategies including traditional breeding,induced mutagenesis,molecular marker-assisted selection and transgenic engineering in the research of a new generation of functional rice that posses health benefits,therapeutic values,bioreactor properties,and other unique functions. We then point out the prospect of functional rice from the point of view of social development,germplasm innovation,breeding of functional rice by biological technology and the test of active material related functional rice. We conclude that developing and marketing of functional rice is becoming a subject of great importance for research and is to be the focus of research in future.

Evaluation of Rice Germplasm for Resistance to the Small Brown Planthopper (Laodelphax striatellus) and Analysis of Resistance Mechanism

One hundred and thirty-eight rice accessions were screened for resistance to the small brown planthopper (SBPH) (Laodelphax striatellus Fallén) by the modified seedbox screening test. Twenty-five rice accessions with different levels of resistance to SBPH were detected, accounting for 18.1% of the total accessions, which included 2 highly resistant, 9 resistant and 14 moderately resistant varieties. Compared with indica rice, japonica rice was more susceptible to SBPH. Antixenosis test, antibiosis test and correlation analysis were performed to elucidate the resistance mechanism. The resistant check Rathu Heenati (RHT), highly resistant varieties Mudgo and Kasalath, and resistant variety IR36 expressed strong antixenosis and antibiosis against SBPH, indicating the close relationship between resistance level and these two resistance mechanisms in the four rice varieties. Antibiosis was the dominant resistance pattern in the resistant varieties Daorenqiao and Yangmaogu due to their high antibiosis but low antixenosis. Dular, ASD7 and Milyang 23 had relatively strong antixenosis and antibiosis, indicating the two resistance mechanisms were significant in these three varieties. The resistant DV85 expressed relatively high level of antixenosis but low antibiosis, whereas Zhaiyeqing 8 and Guiyigu conferred only moderate antibiosis and antixenosis to SBPH, suggesting tolerance in these three varieties. Antibiosis and antixenosis governed the resistance to SBPH in the moderately resistant accession 9311. Antixenosis was the main resistance type in V20A. Tolerance was considered to be an important resistance mechanism in Minghui 63 and Yangjing 9538 due to their poor antibiosis and antixenosis resistance. The above accessions with strong antibiosis or antixenosis were the ideal materials for the resistance breeding.

Genetic Diversity among Parents of Hybrid Rice Based on Cluster Analysis of Morphological Traits and Simple Sequence Repeat Markers

The genetic diversity of 41 parental lines popularized in commercial hybrid rice production in China was studied by using cluster analysis of morphological traits and simple sequence repeat (SSR) markers. Forty-one entries were assigned into two clusters (i.e. early or medium-maturing cluster; medium or late-maturing cluster) and further assigned into six sub-clusters based on morphological trait cluster analysis. The early or medium-maturing cluster was composed of 15 maintainer lines, four early-maturing restorer lines and two thermo-sensitive genic male sterile lines, and the medium or late-maturing cluster included 16 restorer lines and 4 medium or late-maturing maintainer lines. Moreover, the SSR cluster analysis classified 41 entries into two clusters (i.e. maintainer line cluster and restorer line cluster) and seven sub-clusters. The maintainer line cluster consisted of all 19 maintainer lines, two thermo-sensitive genic male sterile lines, while the restorer line cluster was composed of all 20 restorer lines. The SSR analysis fitted better with the pedigree information. From the views on hybrid rice breeding, the results suggested that SSR analysis might be a better method to study the diversity of parental lines in indica hybrid rice.

Identification of QTLs Underlying Folate Content in Milled Rice

Understanding the genetic mechanism underlying folate biosynthesis and accumulation in rice would be beneficial for breeding high folate content varieties as a cost-effective approach to addressing widespread folate deficiency in developing countries. In this study, the inheritance of rice grain folate content was investigated in the Lemont/Teqing recombinant inbred lines and the Koshihikari/Kasalath//Koshihikari backcross inbred lines. 264 F12 recombinant inbred lines(RILs) and 182 BC1F10 backcross inbred lines(BILs) with their parents planted in randomized complete blocks with two replicates in 2010, and RILs harvested in 2008 were used for QTL detection using inclusive composite interval mapping(ICIM) method. In the RIL population, two QTLs, denoted by qQTF-3-1 and qQTF-3-2(QTF, quantitative total folate), explaining 7.8% and 11.1-15.8% of the folate content variation were detected in one or two years, respectively. In the BIL population, a QTL, denoted by qQTF-3-3, was detected, explaining 25.3% of the variation in folate content. All the positive alleles for higher folate content were from the high-folate parents, i.e., Teqing and Kasalath. The known putative folate biosynthesis genes do not underlie the QTLs detected in this study and therefore may be novel loci affecting folate content in milled rice. QTLs identified in this study have potential value for marker assisted breeding for high-folate rice variety.

Cloning and Expression of Gene Responsible for High-Tillering Dwarf Phenotype in Indica Rice Mutant gsor23

High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied byγ-ray.Genetic analysis showed that this phenotype was controlled by one single recessive gene,which was mapped within a physical distance of 386 kb between two insertion-deletion(InDel)markers C1-WT2 and C1-WT4 on the long arm of chromosome 1.There is a known gene D10 within this region,the mutation of which causes high-tillering in rice.Sequence analysis of the D10 allele in gsor23 revealed that the base cytosine(C)at the 404thposition in the coding region was deleted,which would cause frameshift mutation after the 134thamino acids.The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2.Therefore,gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8(CCD8),a key enzyme involved in the biosynthesis of the new plant hormone strigolactones(SLs).After treatment with GR24,a synthetic analogue of SLs,the high-tillering phenotype of gsor23 was restored to normal.Real-time RT-PCR analysis showed that D10 expression was high in roots,but low in leaves.Compared with the wild type Indica9,the expression of the SL biosynthesis gene D10 was upregulated,while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.

Quantitative Trait Loci for Resistance to Stripe Disease in Rice(Oryza sativa)

In order to map the quantitative trait loci for rice stripe resistance, a molecular linkage map was constructed based on the F2 population derived from a cross between Zhaiyeqing 8 and Wuyujing 3. Reactions of the two parents, F1 individual and 129 F2:3 lines to rice stripe were investigated by both artificial inoculation at laboratory and natural infection in the field, and the ratios of disease rating index were scored. The distribution of the ratios of disease rating index in Zhaiyeqing 8/Wuyujing 3 F2:3 population ranged from 0 to 134.08 and from 6.25 to 133.6 under artificial inoculation at laboratory and natural infection in the field, respectively, and showed a marked bias towards resistant parent (Zhaiyeqing 8), indicating that the resistance to rice stripe was controlled by quantitative trait loci (QTL). QTL analysis showed that the QTLs detected by the two inoculation methods were completely different. Only one QTL, qSTV7, was detected under artificial inoculation, at which the Zhaiyeqing 8 allele increased the resistance to rice stripe, while two QTLs, qSTV5 and qSTV1, were detected under natural infection, in which resistant alleles came from Zhaiyeqing 8 and Wuyujing 3, respectively. These results showed that resistant parent Zhaiyeqing 8 carried the alleles associated with the resistance to rice stripe virus and the small brown planthopper, and susceptible parent Wuyujing 3 also carried the resistant allele to rice stripe virus. In comparison with the results previously reported, QTLs detected in the study were new resistant genes to rice stripe disease. This will provide a new resistant resource for avoiding genetic vulnerability for single utilization of the resistant gene Stvb-i.

Genetic Mechanism of Dominant Earliness in Kefeng A,a New Rice Cytoplasmic Male Sterile Line

Kefeng A is an early maturing indica cytoplasmic male sterile (CMS) line of rice. Combinations derived from Kefeng A and late maturing indica restorer lines showed dominant earliness to various extents. To understand the genetic basis of dominant earliness, the genotype of photoperiod-sensitive genes in Kefeng A was analyzed using a complete set of heading time near isogenic lines (NILs) EG0 to EG7, ER, LR, T65, T65Eb, T65Ebm, T65m, NIL(Hd1) and NIL(Hd4). Results indicated that Kefeng A contained two dominant photoperiod-sensitive alleles E1 and Se-1u on E1 and Se-1 loci, respectively, and the genotype of photoperiod-sensitivity genes for heading time in Kefeng A was E1E1e2e2E3E3Se-1uSe-1uEf-1Ef-1. Based on the detected heading time genotype, in combination with the heading time of Kefeng A and the early maturing phenomenon in its derived F1 hybrids, it is speculated that Kefeng A might carry a dominant inhibitor gene Su-E1 for the dominant photoperiod-sensitive gene E1, and a recessive inhibitor gene i-Se-1 for another dominant photoperiod-sensitive gene Se-1. The reason why F1 hybrids from Kefeng A exhibited early maturing was hereby analyzed and the breeding value of dominant earliness related genes in Kefeng A was discussed.

Analysis of QTLs for Flag Leaf Shape and Its Response to Elevated CO_2 in Rice(Oryza sativa)

To understand the responses of flag leaf shape in rice to elevated CO2 environment and their genetic characteristics,quantitative trait loci(QTLs)for flag leaf shape in rice were mapped onto the molecular marker linkage map of chromosome segment substitution lines(CSSLs)derived from a cross between a japonica variety Asominori and an indica variety IR24 under free air carbon dioxide enrichment(FACE,200μmol/mol above current levels)and current CO2 concentration(Ambient,about 370μmol/mol).Three flag-leaf traits,flag-leaf length(LL),width(LW)and the ratio of LL to LW (RLW),were estimated for each CSSL and their parental varieties.The differences in LL,LW and RLW between parents and in LL and LW within IR24 between FACE and Ambient were significant at 1%level.The continuous distributions and transgressive segregations of LL,LW and RLW were also observed in CSSL population,showing that the three traits were quantitatively inherited under both FACE and Ambient.A total of 16 QTLs for the three traits were detected on chromosomes 1,2,3,4,6,8 and 11 with LOD(Log10-likelihood ratio)scores ranging from 3.0 to 6.7.Among them,four QTLs (qLL-6*,qLL-8*,qLW-4*,and qRLW-6*)were commonly detected under both FACE and Ambient.Therefore,based on the different responses to elevated CO2 in comparison with current CO2 level,it can be suggested that the expressions of several QTLs associated with flag-leaf shape in rice could be induced by the high CO2 level.

BRITTLE CULM16(BRITTLE NODE) is required for the formation of secondary cell walls in rice nodes

Plant cell walls constitute the skeletal structures of plant bodies,and thus confer lodging resistance for grain crops.While the basic cell wall synthesis machinery is relatively well established now,our understanding of how the process is regulated remains limited and fragmented.In this study,we report the identification and characterization of the novel rice(Oryza sativa L.)brittle culm16(brittle node;bc16)mutant.The brittle node phenotype of the bc16 mutant appears exclusively at nodes,and resembles the previously reported bc5 mutant.Combined histochemical staining and electron microscopy assays revealed that in the bc16 mutant,the secondary cell wall formation and thickening of node sclerenchyma tissues are seriously affected after heading.Furthermore,cell wall composition assays revealed that the bc16 mutation led to a significant reduction in cellulose and lignin contents.Using a map-based cloning approach,the bc16 locus is mapped to an approximately 1.7-Mb region of chromosome 4.Together,our findings strengthen evidence for discretely spatial differences in the secondary cell wall formation within plant bodies.

15th Anniversary of Journal of Integrative Agriculture(JIA)

The year 2017 marks the 15th anniversary of Journal of Integrative Agriculture(JIA),an official journal of the Chinese Academy of Agricultural Sciences(CAAS).JIA has experienced a successful journey during the past 15 years.The21st century began with great challenges in global climate change,food safety,resource scarcity,and other key

在向袁隆平同志学习交流会上的发言

尊敬的各位领导、各位专家:上午好!非常感谢大会主持人给予这样一个机会来交流向袁隆平同志学习的体会。今天我们大家聚集在这儿,就是来缅怀袁隆平的科学精神,同时交流体会。我有三个体会,向大家汇报。向袁隆平同志学习,我认为第一个就是要学习他勇于创新、敢为人先、不断攀登科学高峰的精神。刚才前面的几位专家都介绍了,袁先生在科学的道路上,往往是把大家认为不可能的科学研究变成可能。