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1. 全基因组关联研究揭示茶树氨基酸含量变异的遗传基础
GUO Ya-fei, LI Dai-li, QIU Hai-ji, ZHANG Xiao-liang, LIU Lin, ZHAO Jing-jing, JIANG De-yuan
Journal of Integrative Agriculture    2023, 22 (11): 3364-3379.   DOI: 10.1016/j.jia.2023.10.002
摘要172)      PDF    收藏

茶是世界上最受欢迎的非酒精饮料之一。游离氨基酸,尤其是茶氨酸,鲜味的主要组成。然而,关于茶树氨基酸含量变异的遗传基础仍不清晰此,基于靶向代谢组学的方法本研究连续两年检测了174份茶树种质嫩叶的游离氨基酸含量,并通过转录组分析获得了这些种质的基因型。基于代谢表型和基因型,通过全基因组关联研究研究影响茶树鲜叶游离氨基酸含量变异的位点。本研究鉴定到69-log10 (P-value) 大于 5位点。功能注释的结果分析显示支链氨基酸转移酶、谷氨酰胺合成酶、硝酸盐转运蛋白和谷氨酸脱羧酶可能在氨基酸代谢的过程发挥重要作用。因此,本研究从中选择了两个显著的位点:谷氨酰胺合成酶Glu1P=3.71×10-4Arg1P=4.61×10-5和支链氨基酸转移酶(Val1P=4.67×10-5I_Leu1P=3.56×10-6CsGSCsBCAT进行基因型分析,选择CsGS的两个等位基因CsGS-LCsGS-HCsBCAT的两个等位基因CsBCAT-LCsBCAT-H进行功能验证。CsGS-LCsGS-H过表达提高了转基因植株中谷氨酸和精氨酸含量CsBCAT-LCsBCAT-H过表达促进了缬氨酸、异亮氨酸和亮氨酸的积累。体外酶活性分析发现SNP1054CsGS催化谷氨酸生成谷氨酰胺的酶活性具有重要影响。此外,CsGS-LCsGS-H差异调控谷氨酰胺积累,CsBCAT-LCsBCAT-H差异调控支链氨基酸积累。综上所述,本研究结果将为茶树氨基酸含量变异的遗传基础解析提供新的认识,并为鉴定优质基因以提高茶树氨基酸含量提供理论依据。

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2. Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection
YAN Zhi-yong, ZHAO Mei-sheng, MA Hua-yu, LIU Ling-zhi, YANG Guang-ling, GENG Chao, TIAN Yan-ping, LI Xiang-dong
Journal of Integrative Agriculture    2021, 20 (7): 1871-1879.   DOI: 10.1016/S2095-3119(20)63275-0
摘要192)      PDF    收藏

番茄褐色皱果病毒(tomato brown rugose fruit virus,ToBRFV)是2015年首次报道的一种烟草花叶病毒属病毒,是番茄安全生产的严重威胁。该病毒已经传播到美洲、亚洲和欧洲的十个国家。2019年,ToBRFV在中国山东发生。本论文旨在明确ToBRFV山东分离物(ToBRFV-SD)的症状、寄主范围和分子特性,并建立一种有效的检测方法。田间调查ToBRFV-SD在不同品种的症状表现。将ToBRFV-SD接种辣椒、本氏烟、马铃薯、茄子、中烟102和50个番茄品种,鉴定其寄主范围。分段克隆ToBRFV-SD基因组片段,并测定其序列;利用BioEdit version 7.2.6比对ToBRFV所有分离物的全基因组序列,分析序列一致率;利用MEGA version 10.1.5构建系统发育树。根据ToBRFV、烟草花叶病毒(tobacco mosaic virus,TMV)、番茄花叶病毒(tomato mosaic virus,ToMV)和番茄斑萎病毒(tomato spotted wilt virus,TSWV)等四种番茄重要病毒基因组的保守区域设计特异性引物,建立四重RT-PCR检测体系。ToBRFV-SD在番茄叶片引起不同程度的花叶和疱斑,在花萼和花梗上引起坏死,在番茄果实上引起畸形、黄斑和褐色皱缩坏死斑。ToBRFV-SD可侵染番茄、辣椒和本氏烟,隐症侵染马铃薯、茄子和烟草品种中烟102。测试的50个番茄品种均不抗ToBRFV-SD。ToBRFV-SD和以色列分离物ToBRFV-IL基因组的核苷酸和氨基酸一致率最高。在基于全基因组序列的系统进化树中,所有ToBRFV分离物聚集到一个分枝,与烟草花叶病毒分枝距离较近。随后,我们建立了四重RT-PCR检测体系,能够通过一个RT-PCR反应,同时检测并区分ToBRFV、TMV、ToMV和TSWV。本研究明确了ToBRFV-SD的症状、寄主范围和分子特性,建立了能区分ToBRFV、TMV、ToMV和TSWV四重RT-PCR检测体系,对指导ToBRFV的早期检测和防控有积极作用。


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3. Expression, regulation and binding affinity of fatty acid-binding protein 2 in Spodoptera litura
WEN Liang, GAO Gui-ping, HUANG Zhi-qiang, ZHENG Si-chun, FENG Qi-li, LIU Lin
Journal of Integrative Agriculture    2020, 19 (6): 1492-1500.   DOI: 10.1016/S2095-3119(20)63167-7
摘要129)      PDF    收藏
Fatty acid-binding proteins (FABPs) are a family of lipid chaperones, which contribute to systemic metabolic regulation through diverse lipid signalings.  In this study, a midgut-specific FABP gene (Slfabp2) was cloned from Spodoptera litura.  RT-PCR and Western blot analysis indicated that RNA and protein levels of SlFABP2 gradually increased and reached a peak at the prepupal stage and maintained a high level during the pupal stage.  The expression of SlFABP2 protein was induced by starvation treatment.  In vitro binding assay revealed that the recombinant SlFABP2 had high affinities of binding long-chain fatty acids, such as palmitic acid, arachidonate and oleic acid.  The results suggest that SlFABP2 may have a unique function that transports intracellular fatty acids and can regulate the metabolism of lipids in metamorphosis.  This work provides experimental clues for understanding the potential function of SlFABP2 in fatty acid metabolism in S. litura.
 
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4.
Long-term fertilization leads to specific PLFA finger-prints in Chinese Hapludults soil
WANG Qi-qi, LIU Ling-ling, LI Yu, QIN Song, WANG Chuan-jie, CAI An-dong, WU Lei, XU Ming-gang, ZHANG Wen-ju
Journal of Integrative Agriculture    2020, 19 (5): 1354-1362.   DOI: 10.1016/S2095-3119(19)62866-2
摘要120)      PDF    收藏
Soil microbes play essential roles in the biogeochemical processes of organic carbon and nutrient cycling.  Many studies have reported various short-term effects of fertilization on soil microbes.  However, less is known about the effects of long-term fertilization regimes on the rhizosphere.  Therefore, the objective of this study was to explore how the soil microbial communities in the rhizosphere respond to different long-term fertilization strategies.  Based on a 21-year field treatment experiment in Guizhou, China, we extracted phospholipid fatty acids (PLFAs) to determine the microbial community structure in both the non-rhizosphere (NR) and rhizosphere (R).  Six treatments were included: no fertilizer (CK), mineral nitrogen fertilizer (N), N with potassium (NK), phosphorus with K (PK), NPK, and NPK combined with manure (MNPK).  The results showed that total PLFAs under unbalanced mineral fertilization (N, NK and PK) were decreased by 45% on average in the NR compared with CK, whereas MNPK increased fungi and G–bacteria abundance significantly in both the NR (by 33 and 23%) and R (by 15 and 20%), respectively.  In addition, all microbial groups in the R under these treatments (N, NK and PK) were significantly increased relative to those in the NR, except for the ratio of F/B and G+/G–, which might be due to the high nutrient availability in the R.  Soil pH and SOC significantly regulated the soil microbial community and structure, explaining 51 and 20% of the variation in the NR, respectively.  However, the rhizosphere microbial community structure was only significantly affected by soil pH (31%).  We concluded that the soil microbial community in the NR was more strongly affected by long-term fertilization than that in the R due to the rhizosphere effect in the agricultural ecosystem.  Rhizosphere nutrient conditions and buffering capacity could help microbial communities resist the change from the long-term fertilization.
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5.
Genome-wide identification and expression analysis of asparagine synthetase family in apple
YUAN Xi-sen, YU Zi-peng, LIU Lin, XU Yang, ZHANG Lei, HAN De-guo, ZHANG Shi-zhong
Journal of Integrative Agriculture    2020, 19 (5): 1261-1273.   DOI: 10.1016/S2095-3119(20)63171-9
摘要106)      PDF    收藏
Asparagine is an efficient nitrogen transport and storage carrier.  Asparagine synthesis occurs by the amination of aspartate which is catalyzed by asparagine synthetase (ASN) in plants.  Complete genome-wide analysis and classifications of the ASN gene family have recently been reported in different plants.  However, systematic analysis and expression profiles of these genes have not been performed in apple (Malus domestica).  Here, a comprehensive bioinformatics approach was applied to identify MdASNs in apple.  Then, plant phylogenetic tree, chromosome location, conserved protein motif, gene structure, and expression pattern of MdASNs were analyzed.  Five members were identified and distributed on 4 chromosomes with conserved GATase-7 and ASN domains.  Expression analysis indicated that all MdASNs mRNA accumulated at the highest level in reproductive organs, namely flowers or fruits, which may be associated with the redistribution of free amino acids in plant metabolic organs and reservoirs.  Additionally, most of MdASNs were dramatically up-regulated under various nitrogen supplies, especially in the aboveground part.  Taken together, MdASNs may be assigned to be responsible for the nitrogen metabolism and asparagine synthesis in apple.
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6. OsHemA gene, encoding glutamyl-tRNA reductase (GluTR) is essential for chlorophyll biosynthesis in rice (Oryza sativa)
ZENG Zhao-qiong, LIN Tian-zi, ZHAO Jie-yu, ZHENG Tian-hui, XU Le-feng, WANG Yi-hua, LIU Ling-long, JIANG Ling, CHEN Sai-hua, WAN Jian-min
Journal of Integrative Agriculture    2020, 19 (3): 612-623.   DOI: 10.1016/S2095-3119(19)62710-3
摘要142)      PDF    收藏
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.
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7. A Dominant Locus, qBSC-1, Controls β Subunit Content of Seed Storage Protein in Soybean (Glycine max (L.) Merri.)
WANG Jun, LIU Lin, GUO Yong, WANG Yong-hui, ZHANG Le, JIN Long-guo, GUAN Rong-xia, LIU Zhang-xiong, WANG Lin-lin, CHANG Ru-zhen , QIU Li-juan
Journal of Integrative Agriculture    2014, 13 (9): 1854-1864.   DOI: 10.1016/S2095-3119(13)60579-1
摘要1422)      PDF    收藏
Soybean seed storage protein is one of the most important plant vegetable proteins, and β subunit is of great significance to enhance soybean protein quality and processing property. F2 segregated population and residual heterozygous lines (RHL) derived from the cross between Yangyandou (low level of β subunit) and Zhonghuang 13 (normal level of β subunit) were used for mapping of β subunit content. Our results showed that β subunit content was controlled by a single dominant locus, qBSC-1 (β subunit content), which was mapped to a region of 11.9 cM on chromosome 20 in F2 population of 85 individuals. This region was narrowed down to 2.5 cM between BARCSOYSSR_20_0997 and BARCSOYSSR_20_0910 in RHL with a larger population size of 246 individuals. There were 48 predicted genes within qBSC-1 region based on the reference genome (Glyma 1.0, Williams 82), including the two copies of β subunit coding gene CG4. An InDel marker developed from a thymine (TT) insertion in one copy of CG4 promoter region in Yangyandou cosegregrated with BARCSOYSSR_20_0975 within qBSC-1 region, suggesting that this InDel marker maybe useful for marker-assisted selection (MAS).
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8. CDH1, a Novel Surface Marker of Spermatogonial Stem Cells in Sheep Testis
ZHANG Yan, WU Sachula, LUO Fen-hua, Baiyinbatu, LIU Lin-hong, HU Tian-yuan, YU Bo-yang, LI Guang-peng , WU Ying-ji
Journal of Integrative Agriculture    2014, 13 (8): 1759-1765.   DOI: 10.1016/S2095-3119(13)60689-9
摘要1493)      PDF    收藏
Spermatogonial stem cells (SSCs) are unique stem cells in adult body that can transmit genetic information to the next generation. They have self-renewal potential and can continuously support spermatogenesis throughout life of a male animal. However, the SSC population is extremely small, isolation and purification of the SSCs is challenging, especially for livestock animals. It has been confirmed that CDH1 (cadherin-1, also known as E-cadherin) can be expressed in undifferentiated SSCs of mouse and rats, but it has not been verified in sheep. Here, CDH1 was found as a novel surface marker for sheep SSCs. In this paper, sheep anti- CDH1 polyclonal antibodies were prepared and its activity was checked. Using the obtained antibodies and immunohistochemistry analysis, we confirmed that CDH1 can be expressed by SSCs in sheep testis.
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9. Variation of Potential Nitrification and Ammonia-Oxidizing Bacterial Community with Plant-Growing Period in Apple Orchard Soil
LIU Ling-zhi, QIN Si-jun, Lü De-guo, WANG Bing-ying , YANG Ze-yuan
Journal of Integrative Agriculture    2014, 13 (2): 415-425.   DOI: 10.1016/S2095-3119(13)60424-4
摘要1782)      PDF    收藏
In this study, we investigated the potential nitrification and community structure of soil-based ammonia-oxidizing bacteria (AOB) in apple orchard soil during different growth periods and explored the effects of environmental factors on nitrification activity and AOB community composition in the soil of a Hanfu apple orchard, using a culture-dependent technique and denaturing gradient gel electrophoresis (DGGE). We observed that nitrification activity and AOB abundance were the highest in November, lower in May, and the lowest in July. The results of statistical analysis indicated that total nitrogen (N) content, NH4 +-N content, NO3 --N content, and pH showed significant correlations with AOB abundance and nitrification activity in soil. The Shannon-Winner diversity, as well as species richness and evenness indices (determined by PCR-DGGE banding patterns) in soil samples were the highest in September, but the lowest in July, when compared to additional sampled dates. The DGGE fingerprints of soil-based 16S rRNA genes in November were apparently distinct from those observed in May, July, and September, possessing the lowest species richness indices and the highest dominance indices among all four growth periods. Fourteen DGGE bands were excised for sequencing. The resulting analysis indicated that all AOB communities belonged to the β-Proteobacteria phylum, with the dominant AOB showing high similarity to the Nitrosospira genus. Therefore, soil-based environmental factors, such as pH variation and content of NH4 +-N and NO3 --N, can substantially influence the abundance of AOB communities in soil, and play a critical role in soil-based nitrification kinetics.
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10. A Novel Approach to the Water Uptake Dynamics in Roots of Maize, Wheat and Barley Under Salt Stress
BU Qing-mei, BIAN Dian-xia, LIU Lin-de , ZHU Jian-jun
Journal of Integrative Agriculture    2012, 12 (4): 576-584.   DOI: 10.1016/S1671-2927(00)8577
摘要1564)      PDF    收藏
The water uptake dynamics in maize, wheat, and barley under salt stress were investigated with a xylem pressure probe. The average xylem pressure responses to salt stress in the three plants were 36, 93, and 89% of the osmotic stresses for maize, wheat, and barley, respectively, which are significantly smaller than the magnitude of the osmotic stresses being applied. In order to explain the thermodynamic discrepancies among the water potential changes in the root xylem of the three plants, a novel approach, tentatively named the “symplastic flow dilution model” was proposed in this paper. The model was presented in an attempt to give answers to the problem of how the roots under salt stress could absorb water when the water potential of the xylem sap is considerably higher than that of the solution in the root ambient. According to the model, the salt solution in the microenvironment of the endodermis of a root was diluted to some extent by the efflux from cells so the central stele of the root is not exposed to the same solution bathing the root with the same salt concentration. In contrast, we also presented another approach, the “reflection coefficient progression approach”, which was less likely to be true because it requires a considerable amount of solute to be transported into the root xylem when the salt stress is severe.
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