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10 November 2016, Volume 30 Issue 6 Previous Issue    Next Issue
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Response of Gene Expression Related to Efficient Phosphorus Absorption and Utilization to Low-P Stress in Rice Roots Xun-xia HU, Chun-yang SHI, Yan DING, Ping ZHANG, Yong-sheng GE, Yu-jin LIU, Ze-gang WANG, Cai-lin GE 2016, 30(6): 567-576.  DOI: 10.16819/j.1001-7216.2016.6017 Abstract ( )   HTML ( )   PDF (1264KB) ( )   It is important to elucidate the mechanism of low phosphorus tolerance in rice for the improvement of utilization efficiency of soil phosphorus. With two different rice varieties, low-P tolerant variety Yi 2434 and low-P sensitive variety Tongjing 981, the gene expression related to phosphorus absorption and utilization in rice seedling roots were determined by using gene chip and qRT-PCR technology. The phosphorus content showed that compared with the control group, the phosphorus content in the leaves of Yi 2434 decreased by a less percentage than that in Tongjing 981 under low-P stress, indicating that Yi 2434 had a high efficiency in phosphorus absorption from low phosphorus medium compared with Tongjing 981. The gene chip results showed that the expressions of PHR1, osa-miR399s and SPXs triggered the phosphorus starvation signal pathway, and the induced gene expressions of APA, PAPs, MPE, PA, PEPC and VDAC1, C4-DT/MAT enhanced the synthesis and secretion of organophosphorus hydrolase and organic acid, promoted the reactivation of insoluble phosphorus in media, and the induced gene expressions of OsPT2 and OsPT6 accelerated the absorption of phosphorus in Yi 2434 roots under low-P stress. qRT-PCR results showed that the transcriptional levels of the eight key genes(PHR1,SPX,PAP,APA,PEPC,MFS,OsPT2,OsPT6 ) related to phosphorus starvation signal transduction, phosphorus activation and efficient absorption were continuously increased along with the low-P treatment in Yi 2434 roots. Furthermore, the transcriptional levels of these eight genes in Yi 2434 roots were significantly higher than those in Tongjing 981 roots under the low-P stress and the induced expression of PHR1, APA, OsPT2 genes in Tongjing 981 roots was very low under low-P stress. Additionally, the activities of both acid phosphatase (APA) in the tissues of roots and secreted by roots in Yi 2434 increased more significantly than that in Tongjing 981, which is possibly one of the key pathway to confer Yi 2434 higher tolerance to the low-P stress than Tongjing 981.

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CRISPR/Cas9 System-based Editing of OsbHLH116 Gene and Its Off-target Effect Analysis Yan-xiu DU, Xin JI, Hui-jie CHEN, Ting PENG, Jing ZHANG, Jun-zhou LI, Hong-zheng SUN, Quan-zhi ZHAO 2016, 30(6): 577-586.  DOI: 10.16819/j.1001-7216.2016.6024 Abstract ( )   HTML ( )   PDF (2684KB) ( )   With OsbHLH116 as the editable object, the 19-bp sgRNA was designed in the exon 1 site according to the coding sequence (CDS) and the oligonucleotides of sgRNA were chemically synthesized and inserted into linearized plasmid pBUN411. The transgenic lines were obtained by Agrobacterium-mediated transformation method. Identification of mutants and off-target effects analysis of OsbHLH116 were conducted by combing restriction enzyme digestion with sequencing. The results showed that the recombinant vector of pBUN411-gRNA succeeded in oriented editing of OsbHLH116. The restriction enzyme analysis results indicated that we got 6 mutants from 10 randomly selected transgenic lines. Sequence analysis of the six mutants indicated that homozygous mutation, biallelic mutation and heterozygous mutation occured. After searching the rice genome using the target sequence with PAM, two highly identical sites were found. However, we did not observe any mutation at these sites by restriction enzyme.

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Cloning and Characterization of a Small Heat Shock Protein (SHSP) Gene in Rice Plant Cong-ying XIANG, Nian-jun CAI, Jing LI, Jian YANG, Jian-ping CHEN, Heng-mu ZHANG 2016, 30(6): 587-592.  DOI: 10.16819/j.1001-7216.2016.6056 Abstract ( )   HTML ( )   PDF (3349KB) ( )   Small heat shock proteins (SHSPs) exist widely in all kinds of creatures and play an important role in both development and stress responses. A gene encoding a SHSP (OsSHSP17.6) was cloned from Oryza sativa and the sequence assembly showed the encoding region of OsSHSP17.6 was 477 bp in length, encoding a polypeptide of 158 aa with a molecular weight of 17.6 kD. Sequence analysis showed that such kind of SHSPs was conserved in different plants and belonged to the cytosolic class I (CI). The transcriptional level of OsSHSP17.6 was significantly up-regulated by heat shock with qRT-PCR. The protein appeared to be accumulated up to higher level under the condition of heat shock in western blotting assays. Taken together, these findings consistently indicated that OsSHPS17.6 could be involved into the response to heat shock, which could contribute to understanding the function of OsSHSP17.6.

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Research on DNA Molecular Digital Fingerprint Database Based on 48 Pairs of SSR Primers for 94 Hybrid Rice Parents in NYT 1433-2014 LIN Yixia, WANG Zixin, LIU Huan, WANG Zheng, LIANG Manzhong, DAI Xiaojun*, CHEN Liangbi* 2016, 30(6): 593-602.  DOI: 10.16819/j.1001-7216.2016.6028 Abstract ( )   HTML ( )   PDF (1580KB) ( )   It is of great significance to establish the simple molecular fingerprinting technique with high resolution for identification of the genetic polymorphism and the authenticity of different rice varieties, so as to guide rice breeding and regulate its seed market. The new standards of technical regulation on identification of the rice varieties with SSR markers, formulated by the Ministry of Agriculture of P.R. China, recommended the use of 35 control standard reference samples with different genetic characteristics for identification of rice varieties. The genetic polymorphism and specificity of 94 hybrid rice parents were compared based on the standard method. The results indicated that the tested varieties differed at least three pairs of mutated loci or the genetic differences between the parents of hybrid rice could been well distinguished. By comparing the polymorphism of the 48 recommended primers of the new standard, 46 primers showed higher polymorphism except RM176 and RM551. Thus, higher polymorphic alternative molecular markers would be identified in other loci of the same chromosome. 16 new allelic variation sites were found and  could be used as supplementary of standard fingerprint database and enrich genetic variation sites information. By analyzing molecular fingerprint of 94 hybrid parental materials, 23 have specific molecular markers and can be used in authenticity analysis of hybrid combination and purity identification of hybrid seeds. According to the digital molecular fingerprint of the tested rice parent, we constructed a virtual digital molecular fingerprint database including 87 female sterile lines and 7 male parents, and specific digital molecular marker of virtual hybridized combination authenticity and rapid seed purity identification.

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Genetic Analysis and Gene Mapping of a Pale Green Leaf Mutant HM133 in Rice Yong-feng SHI, Yan HE, Dan GUO, Xiang-guang LV, Qi-na HUANG, Jian-li WU 2016, 30(6): 603-610.  DOI: 10.16819/j.1001-7216.2016.6033 Abstract ( )   HTML ( )   PDF (1492KB) ( )   The pale green leaf mutant HM133 was identified from an EMS-induced IR64 mutant bank. The contents of photosynthetic pigments including chlorophyll and carotenoid of HM133 were reduced significantly at 6 weeks and 15 weeks after sowing when compared with IR64.The net photosynthetic rate of HM133 was considerably lower than that of the wild-type IR64 at heading stage while the stomatal conductance was apparently increased. The agronomic traits including plant height, number of filled grain per panicle and seed-setting rate decreased significantly in the mutant compared with the wild-type. In addition, the mutant exhibited a less number of grana, irregular arrangement of thylakoid layer in the chloroplast at the tillering stage. Genetic and mapping analysis showed that the pale green phenotype was controlled by a single recessive gene located in the long arm of chromosome 3 between SSR markers RM143 and RM3684. The interval contains an ORF OsChlD encoding magnesium-chelatase D subunit. Sequence analysis revealed that the mutant allele carried a nucleotide substitution from G to A in the tenth exon of OsChlD, which led to the substitution of glutamic acid for arginine acid. Therefore, it is deduced that OsChlD is the candidate gene controlling the pale green leaf phenotype of HM133.

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Effect of Ordered Transplanting and Optimized Broadcasting on Rice Root System Morphological and Physiological Characteristics Under Super High-yielding Cultivation Bao-wei GUO, Ke XU, Hong-cheng ZHANG, Qi-gen DAI, Zhong-yang HUO, Hai-yan WEI, Hou-cun CHEN 2016, 30(6): 611-625.  DOI: 10.16819/j.1001-7216.2016.6074 Abstract ( )   HTML ( )   PDF (1244KB) ( )   The rice root system morphological and physiological characteristics were to explore the approaches to achieve super high yield. Super rice Wuyunjing 24 and Nanjing 44 were used as experimental material and three planting methods including ordered transplanting (OT), optimized broadcasting (OB) and cast transplanting(CT) were designed to investigate the root system morphological and physiological characteristics with mechanical transplanting(MT) as control. The results were as follows: root length, number and weight of 3-hole seedlings had obvious advantages 7 d after transplanting and 2-hole seedlings had obvious advantages 15 d after transplanting. Root dry weight, root shoot ratio and activity of root system at each stage followed the trend of OT>OB>CT>MT, while treatments of different holes showed a trend of 2-hole>3-hole>1-hole. And total root absorbing surface area, active absorbing area, ratio of active absorbing surface area to total absorbing surface area and root bleeding sap showed the same trend. Root dry weight, root shoot ratio, root activity, total absorbing surface area, active absorbing area, ratio of active absorbing surface area to total absorbing surface area and root bleeding sap had a very significant positive correlation with grain yield. Root at the depth of top 5 cm accounted for more than 70%, while at depth of top 10 cm accounted for more than 90%. And root dry weight, root volume and root weight density at each layer showed the trend of OT>OB>CT, 2-hole>3-hole >1-hole. Root weight ratio at the depth of 5-10 cm, 10-15 cm and 15-20 cm showed the same trend. And root dry weight at each layer was very significantly correlated with grain yield, and root at the upper layer made greater contributions to the grain yield, and the contribution rate of root to yield at the top 10 cm depth was more than 90%. And it could be concluded that, ordered transplanting and optimized broadcasting rice with 2-hole seedling had quicker root growth, higher root activities and more reasonable root distribution at late growing stage which were the physiological basis for super high yield.

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Effects of Root-cutting Treatment on Growth and Physiological Characteristics of Hybrid Rice at Different Seedling-ages Ying-hong LI, Hai-yue WANG, Feng-jun YAN, Na LI, Yong-jian SUN, Zou DAI, Hua-ying XIE, Jun MA 2016, 30(6): 626-636.  DOI: 10.16819/j.1001-7216.2016.6057 Abstract ( )   HTML ( )   PDF (2518KB) ( )   Supper hybrid rice F you 498 was used to investigate the effects of seedling-age (25 days old and 40 days old) and root-cutting (1/3, 2/3 and the whole root were cut off with no root-cutting as control) on the root response 0-48 hours and 1-29 days after root cutting, as well as dynamics of biomass accumulation, root morphology and nitrogen (N) absorption in a hydroculture experiment. The results showed that rice seedlings at various seedling ages responsed diversely to root-cutting. The proline content and soluble sugar content of the above-ground part had a significant peak after root-cutting, which was higher and reached the peak value in shorter time with the increase of the root-cutting ratio. The physiological resistance and resilience after root-cutting of 25-day seedling-age were superior to that of 40-day old seedling. In the views of root-cutting ratio, 1/3 root-cutting had positive effect on seedling growth. Compared to the control, the root length, root surface area, above-ground biomass, root biomass, nitrogen accumulation amount and root shoot ratio were higher 22 days after 1/3 root-cutting for 25-day old seedling. However, the root surface area in the whole growth stage decreased with the increaing root-cutting ratio for 40-day old seedling. The root-shoot ratio was higher than that of control 22 days after 1/3 root-cutting, and the above-ground biomass, root length, nitrogen accumulation amount and nitrogen content were also higher than those of control 29 days after 1/3 root-cutting. Besides, the root damage might inhibite the seedling growth and restrain the absorption of nutrients, which could affect the growth and development of rice seedling.

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Effect of High Temperature Stress on Physiological Characteristics of Spikelet of Rice During Flowering Stage Xu LAN, Zheng-dong GU, Yan-fei DING, Ke WANG, Qiong JIANG, Cheng* ZHU 2016, 30(6): 637-646.  DOI: 10.16819/j.1001-7216.2016.5178 Abstract ( )   HTML ( )   PDF (1410KB) ( )   To ascertain the effect of high temperature stress on the physiological characteristics of rice spikelet at flowing stage, wild type Zhonghua 11(ZH11) and its heat-resistant mutant (hst) were exposed to heat stress (40℃,6 h per day) for 7 days in growth chambers. The results showed that under heat stress the chlorophyll content in flag leaf, soluble protein and soluble sugar contents in spikelet of rice decreased obviously with lower decline in the mutant hst compared to the wild type ZH11. While malonyldialdehyde (MDA), hydrogen peroxide(H2O2) and free proline contents in spikelets increased significantly with greater increase in wild type ZH11 than in the mutant hst. Superoxide dismutase (SOD), catalase (CAT) and peroxidase(POD) activities showed different responses to high temperature stress, and the correlation between the enzyme activities and the thermal sensitivity would be further investigated. Under high temperature stress, higher photosynthesis efficiency, better osmotic adjustment ability as well as lower MDA and H2O2 content of spikelets in the mutant could be the physiological basis of high temperature tolerance.

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Impacts of Rice-Fish Co-culture on the Physical and Chemical Variables of the Microprofiles at Sediments-Water Interface in an Intensive-culture Pond Lin QIN, Yao-bin LIU, Feng-bo LI, Jin-fei FENG, Dian-xing WU, Fu-ping FANG 2016, 30(6): 647-652.  DOI: 10.16819/j.1001-7216.2016.6077 Abstract ( )   HTML ( )   PDF (851KB) ( )   An experiment was carried out to investigate the physical and chemical variables in sediment-water interface of yellow catfish pond with and without rice planting. The results showed that rice-fish co-cultures slowed down the decrease of O2 concentration around the sediment-water interface, and increase the O2 penetration depths by 71.4%, comparing with fish monoculture pond. Additionally, rice-fish co-culture increased the Eh value, and improved the aerobic condition in the sediment of pond. Rice-fish co-culture also decreased the pH around sediment-water interface. The pH of rice-fish co-culture pond was lower than that of monoculture pond by one unit. These results indicated that rice growing changed the mocroenvironment of the sediment-water interface of fish pond, which may affect the migration and transformation of elements around this interface.

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Molecular Cloning and Functional Analysis of NlTgo in the Rice Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) Long-fei CHEN, Pin-jun WAN, Wei-xia WANG, Qiang FU, Ting-heng ZHU 2016, 30(6): 653-660.  DOI: 10.16819/j.1001-7216.2016.6032 Abstract ( )   HTML ( )   PDF (2123KB) ( )   Transcriptional regulator Tango (Tgo) plays crucial roles in insect development, including neurogenesis, hematopoiesis, sex determination and gut development. In this study, a full-length cDNA of Tango was cloned in Nilaparvata lugens. qRT-PCR and RNA interference (RNAi) were further used to analyze the expression pattern and function role, respectively. Our results showed that NlTgo contained a 2007-bp open reading frame (ORF), encoding 669 amino acid residues. Sequence alignment showed that NlTgo shared an identity of 68% with Pediculus humanus. Phylogenetic analysis suggested that NlTgo was closely related to the Tango proteins from Acyrthosiphon pisum, Pyrrhocoris apterus and Cimex lectularius. Expression profile revealed that NlTgo expression was higher in the first- and second-instar larvae than that in eggs. Furthermore, the expression level of NlTgo was higher in ovary than that in integument. Knocking down of NlTgo, in the 4th-instar nymph, was performed by double-stranded RNA (dsRNA) targeting NlTgo. It was found that the expression level of NlTgo, 4 days after injection, was significantly decreased by 77%, compared with control. Furthermore, nymphs died due to the abnormal molting, and the survival rate was only 23%, significantly lower than control group (98%). The results suggest that NlTgo is involved in the development of N.lugens and can serve as a potential target for controlling the brown planthopper.

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Development of Functional Marker for Rice stripe virus Resistant Gene STV11 and Resistant Germplasm Selection in japonica Rice Ting MAO, Xu LI, Zhan ZHANG, Li-dong FU, Zhen-yu LI 2016, 30(6): 661-667.  DOI: 10.16819/j.1001-7216.2016.6058 Abstract ( )   HTML ( )   PDF (1419KB) ( )   The Rice stripe virus (RSV) causes one of the most serious viral diseases of rice (Oryza sativa L.). STV11 is a rice stripe virus resistant gene derived from india rice Kasalath. We developed a simple PCR-based functional marker named qstv11 which targets the functional six bp delection polymorphism at STV11KAS. The genotype on STV11 and rice stripe virus resistance of 299 varieties was analyzed, including five resistant varieties, five susceptible varieties, 41 varieties from Liaoning Province, 17 Japanese japonica varieties, 142 recombinant inbred lines (Akihikari/Qishanzhan,population A), 85 backcross recombinant inbred lines (Sasanishiki/Habataki//Sasanishiki population,B) and 4 parents. The newly developed functional marker was highly efficient in discriminating resistant genotypes from susceptible ones and showed perfect co-segregation with the trait of rice stripe virus resistance in the test varieties. One variety “Kangyan 100” was identified with STV11-R from Liaoning varieties. All the varieties from Japanese japonica varieties were susceptible to rice stripe virus. Twenty four and six varieties were identified with STV-R from population A and population B, respectively. Several varieties with STV11-R type with japonica background were detected.

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Observation of Sexual Structure of Magnaporthe oryzae via Calcofluor White and Nile Red Staining Zhuo-kan GU, Ling LI, Jiao-yu WANG, Rong-yao CHAI, Yan-li WANG, Zhen ZHANG, Xue-qin MAO, Hai-ping QIU, Guo-chang SUN 2016, 30(6): 668-672.  DOI: 10.16819/j.1001-7216.2016.6035 Abstract ( )   HTML ( )   PDF (979KB) ( )   Magnaporthe oryzae is an economically important heterothallic ascomycete which causes rice blast, the most destructive rice disease worldwide. To the present, sexual generation of the fungus has been relatively less investigated. In the present work, Guy-11 and 2539, two M. oryzae strains in opposite mating types, were cross cultured on six deferent substrates to induce the sexual generation. The strains produced ascocarps on all of the substrates tested, however, the number, forming time and ripeness degree of the ascocarps varied largely with the producing substrates, among which oatmeal agar(OMA) gave the optimum value. In order to better detect the structures of sexual generation, the asci and ascospores were stained with Fluorescent Brightener and Nile red, and examined using a fluorescence microscopy. Under the fluorescence microscope, the cell walls were stained bright blue by Fluorescent Brightener, which greatly improved the visibility of asci and ascospores. Numbers of asci were found in mature ascocarps, with eight ascospores in each ascus, and an ascospore usually has four cells. Meanwhile, the ascospores can be stained bright orangey-red by Nile red, indicating they are rich in lipids. These findings showed that the fluorescence staining is an effective method to observe the structure of sexual generation in M. oryzae.