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1. 新型18β-甘草次酸酰胺衍生物在控制植物细菌性病害中的双重作用：基于ROS介导的抗菌活性和激活植物防御响应

SONG Ying-lian, LIU Hong-wu, YANG Yi-hong, HE Jing-jing, YANG Bin-xin, YANG Lin-li, ZHOU Xiang, LIU Li-wei, WANG Pei-yi, YANG Song

Journal of Integrative Agriculture 2023, 22 (9): 2759-2771. DOI: 10.1016/j.jia.2022.10.009

摘要（205）

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长期以来，天然产物为新型农用化学品的发现提供了重要的来源及灵感。在众多的天然产物中，18β-甘草次酸具有广谱的生物活性，更为新药发现提供了重要的活性骨架。为了拓展18β-甘草次酸的农业用途，制备了一系列新型18β-甘草次酸酰胺衍生物，并评价其抗菌性能。以水稻黄单胞菌、柑橘溃疡病菌、猕猴桃溃疡病菌为测试菌株，采用浊度法测试了目标化合物的离体抗菌活性；采用盆栽实验，评估了目标化合物对水稻白叶枯病的防治效果；采用透射电镜对细菌的表型进行了初步验证；借助活性氧实验验证化合物对病原菌活性氧的干扰效果；结合防御酶实验探究化合物对植物防御酶活性的调控性能。通过抗菌活性实验表明，化合物5k对水稻白叶枯病菌(Xoo)具有较好的离体抑菌活性(EC₅₀ = 3.64 mg L^-1)和优异的活体保护活性(54.68%)。进一步通过活性氧实验和表型验证，化合物5k能造成病原菌体内活性氧的过量产生和积累，并进一步破坏病原菌的细胞膜。更值得注意的是,化合物5k能提升包括过氧化氢酶、超氧化物歧化酶、过氧化物酶和苯丙氨酸解氨酶在内的植物防御酶的活性。实验结果表明，制备的18β-甘草次酸酰胺衍生物能通过破坏病原菌的氧化还原平衡并激活植物的防御系统共同发挥其控制植物细菌性病害的潜力。

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2. JIA-2021-1010 水平转移转录因子通过调控细胞色素bd氧化酶基因参与Xoc氧胁迫适应性和毒力机制的研究

WANG Pei-hong, WANG Sai, NIE Wen-han, WU Yan, Iftikhar AHMAD, Ayizekeranmu YIMING, HUANG Jin, CHEN Gong-you, ZHU Bo

Journal of Integrative Agriculture 2022, 21 (6): 1673-1682. DOI: 10.1016/S2095-3119(21)63801-7

摘要（392）

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早在2011年，研究者已对其进行了全基因组测序，并发现此基因组中有超过30%的编码基因是假定基因。另外，水平基因转移(horizontal gene transfer，HGT)已被认为是细菌基因组创新和进化的驱动力之一。前人在Xoc应对氧化应激的分子机制研究中，鉴定到了一个参与BLS256响应氧化胁迫并对毒性有贡献的水平转移基因(xoc_2868)。然而，xoc_2868作为一个转录因子的未知编码基因，其调控机制尚未被揭示。本研究基于BLAST序列比对和系统发育分析，初步判断其下游基因(xoc_2866和xoc_2867)与xoc_2868一样，均可能是BLS256在长期进化过程中通过水平转移从伯克氏菌科(Burkholderiaceae)获得的。为探索xoc_2868在BLS256响应氧胁迫过程中的潜在作用，我们分别对野生型(BLS256)和突变株(Δxoc_2868)在氧胁迫处理后进行了转录组测序。RNA-seq数据分析表明，在氧胁迫条件下，突变株中几个参与胞外多糖(EPS)和黄原胶(xanthan)生物合成基因的表达相较野生型显著下调，但未检测到其下游基因(xoc_2866和xoc_2867)的表达。为进一步鉴定受XOC_2868直接调控的基因，我们在野生型xoc_2868 C端融合了His6标签，并对此重组菌株进行了染色质免疫共沉淀 (ChIP-seq)分析。结合转录组分析发现，XOC_2868直接调控一个编码细胞色素bd氧化酶的两个亚基并参与氧化还原平衡的操纵子(cydAB)。与野生型菌株相比，cydA和cydAB缺失突变菌株与Δxoc_2868菌株一致出现对外源H₂O₂敏感性增强和细菌毒力减弱的表型。综上所述，本研究探讨了一种HGT形成和选择驱动的调控回路进化的可能性，xoc_2868与其两个下游基因可能是作为一个基因簇转移的，但它们在BLS256中各自进化，并在外界选择压下得以保留，XOC_2868通过结合新的调控位点直接调控了细胞色素bd氧化酶表达的通路，通过清除H₂O₂和其他ROS保护细胞免受氧化应激。此外，对胞外多糖和黄原胶合成相关基因的间接激活，也促进了其在宿主体内的定植和传播，从而参与了BLS256的致病力。本研究结果强调了在BLS256进化过程中，HGT现象对其毒力和适应性影响的可能性。

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3. Nudix水解酶家族在葡萄果实发育和逆境响应中的多种生理功能分析

WANG Pei-pei, WANG Zhao-ke, GUAN Le, Muhammad Salman HAIDER, Maazullah NASIM, YUAN Yong-bing, LIU Geng-sen, LENG Xiang-peng

Journal of Integrative Agriculture 2022, 21 (1): 91-112. DOI: 10.1016/S2095-3119(20)63490-6

摘要（313）

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Nudix水解酶家族普遍存在于真核和原核生物中，能够水解多种有机焦磷酸盐。尽管Nudix水解酶家族能够清除和调节细胞内产生的有害代谢产物，应答多种非生物和生物胁迫，但葡萄中的Nudix水解酶的功能还未见报道。本研究通过生物信息学分析，在葡萄中鉴定了25个葡萄VvNUDX基因家族成员，并根据其对底物的偏好性将其分为8个亚家族。VvNUDX基因家族在进化过程中存在串联复制和片段复制两种方式。为了研究VvNUDX基因在葡萄生长发育以及胁迫应该过程中的功能，我们利用NCBI公共数据库资源分析这些基因的表达模式。在不同葡萄组织中的时空表达分析表明，VvNUDX基因可能涉及多个生长发育过程。转录组数据和定量PCR分别表明，有10个VvNUDX基因在葡萄果实发育过程中特异表达，表明VvNUDX基因可能在葡萄果实发育过程中扮演重要角色。系统发育和表达分析表明，VvNUDX1和VvNUDX3可能参与葡萄萜类化合物的生物合成。多个ADP-核糖/NADH亚组的VvNUDX基因可以应答多种非生物和生物胁迫，表明VvNUDX基因参与葡萄对非生物和生物胁迫的解毒过程，并调节疾病免疫和抗性途径。相关研究结果为有助于进一步解析VvNUDX基因在葡萄果实发育和胁迫应答网络中的生理功能。

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4. Horizontal gene transfer of a syp homolog contributes to the virulence of Burkholderia glumae

WANG Sai, WANG Pei-hong, NIE Wen-han, CUI Zhou-qi, LI Hong-yu, WU Yan, Ayizekeranmu YIMING, FU Luo-yi, Iftikhar AHMAD, CHEN Gong-you, ZHU Bo

Journal of Integrative Agriculture 2021, 20 (12): 3222-3229. DOI: 10.1016/S2095-3119(20)63553-5

摘要（176）

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水稻细菌性穗枯病又称水稻细菌性谷枯病，是一种由颖壳伯克氏菌 (Burkholderia glumae) 引起的严重的水稻种传病害，对全球水稻生产和食品安全造成了巨大威胁。由于缺乏对B. glumae在植物宿主中的适应性和发病机制的深入了解，迄今生产上还没有有效的防治措施。水平基因转移 (HGT) 已被证明是原核生物进化的主要驱动力。先前对60个Burkholderia全基因组的比较分析推断，大多数Burkholderia基因在其进化过程中至少经历过一次HGT，并在其菌株分化和致病性决定因素中起着重要作用。在本研究中，我们通过对LMG 2196菌株进行全基因组分析，鉴定到了42个潜在的水平转移基因。其中，一个注释为非核糖体肽合成酶(KS03_RS09665)的基因被确定为候选基因。进一步通过系统进化树的建立，发现该基因仅出现在与植物致病相关的Burkholderia菌属，并且在进化分枝上更接近于假单胞菌(Pseudomonas)中编码丁香肽合成酶(SypA)的sypA基因。为研究该基因在B. glumae致病性中的潜在作用，我们构建了syp基因缺失突变株。表型观察结果表明，sypA基因参与调控了该病菌的游动性、生物膜的形成、类似丁香肽代谢物的合成和致病性等重要生理表型。其中，与野生型菌株接种稻穗相比，sypA突变体接种稻穗后发病指数降低了20%。另外，与野生型菌株相比，sypA缺失突变菌株表现为游动能力显著下降、生物膜形成和类似丁香肽代谢物的合成受到抑制。综上所述，本研究探讨了水平转移基因sypA在颖壳伯克氏菌毒力中的作用。结果表明，sypA基因可能参与了颖壳伯克氏菌毒性物质丁香肽的合成，并且正向调控了其游动性和生物膜的形成，从而参与了颖壳伯克氏菌的致病力。本研究的结果强调了在颖壳伯克氏菌进化过程中，HGT现象对其毒力和适应性影响的可能性。

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5. Inheritance of steroidal glycoalkaloids in potato tuber flesh

PENG Zhen, WANG Pei, TANG Die, SHANG Yi, LI Can-hui, HUANG San-wen, ZHANG Chun-zhi

Journal of Integrative Agriculture 2019, 18 (10): 2255-2263. DOI: 10.1016/S2095-3119(19)62718-8

摘要（154）

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Potato (Solanum tuberosum L.) is the third most important food crop worldwide after wheat and rice in terms of human consumption. A critical domestication trait for potato was the decrease of toxic steroidal glycoalkaloids (SGAs) in tuber flesh. Here, we used a diploid F2 segregating population derived from a cross between S. tuberosum and the wild potato species Solanum chacoense to map the quantitative trait loci (QTLs) associated with the regulation of SGAs content in tuber flesh. In a three-year study, we identified two QTLs on chromosomes 2 and 8 affecting SGAs content in tuber flesh. The QTL on chromosome 8 harbors 38 genes that are co-expressed with the GLYCOALKALOID METABOLISM genes. These findings lay the foundation for exploiting the genes controlling SGAs content in tuber flesh and they provide a theoretical basis for the use of wild germplasm in potato breeding.

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6. No-tillage effects on grain yield and nitrogen requirements in hybrid rice transplanted with single seedlings: Results of a long-term experiment

REN Ai-xia, SUN Min, WANG Pei-ru, XUE Ling-zhu, LEI Miao-miao, XUE Jian-fu, GAO Zhi-qiang, YANG Zhen-ping

Journal of Integrative Agriculture 2019, 18 (1): 33-42. DOI: 10.1016/S2095-3119(18)61980-X

摘要（289）

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Sowing date and seeding rate are critical for productivity of winter wheat (Triticum aestivum L.). A three-year field experiment was conducted with three sowing dates (20 September (SD1), 1 October (SD2), and 10 October (SD3)) and three seeding rates (SR67.5, SR90, and SR112.5) to determine suitable sowing date and seeding rate for high wheat yield. A large seasonal variation in accumulated temperature from sowing to winter dormancy was observed among three growing seasons. Suitable sowing dates for strong seedlings before winter varied with the seasons, that was SD2 in 2012–2013, SD3 in 2013–2014, and SD2 as well as SD1 in 2014–2015. Seasonal variation in precipitation during summer fallow also had substantial effects on soil water storage, and consequently influenced grain yield through soil water consumption from winter dormancy to maturity stages. Lower consumption of soil water from winter dormancy to booting stages could make more water available for productive growth from anthesis to maturity stages, leading to higher grain yield. SD2 combined with SR90 had the lowest soil water consumption from winter dormancy to booting stages in 2012–2013 and 2014–2015; while in 2013–2014, it was close to that with SR67.5 or SR112.5. For productive growth from anthesis to maturity stages, SD2 with SR90 had the highest soil water consumption in all three seasons. The highest water consumption in the productive growth period resulted in the best grain yield in both low and high rainfall years. Ear number largely contributed to the seasonal variation in grain yield, while grain number per ear and 1 000-grain weight also contributed to grain yield, especially when soil water storage was high. Our results indicate that sowing date and seeding rate affect grain yield through seedling development before winter and also affect soil water consumption in different growth periods. By selecting the suitable sowing date (1 October) in combination with the proper seeding rate of 90 kg ha^–1, the best yield was achieved. Based on these results, we recommend that the current sowing date be delayed from 22 or 23 September to 1 October.

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7. The PhoR/PhoP two-component system regulates fengycin production in Bacillus subtilis NCD-2 under low-phosphate conditions

GUO Qing-gang, DONG Li-hong, WANG Pei-pei, LI She-zeng, ZHAO Wei-song, LU Xiu-yun, ZHANG Xiao-yun, MA Ping

Journal of Integrative Agriculture 2018, 17 (01): 149-157. DOI: 10.1016/S2095-3119(17)61669-1

摘要（717）

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Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator phoP and its sensor kinase PhoR compose a two-component system in B. subtilis. In this study, the phoR- and phoP-knockout mutants were constructed by in-frame deletion and the role of PhoR/phoP on the production of fengycin was determined. Inactivation of phoR or phoP in B. subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type. The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate, and the lipopeptides from phoR-null mutant or phoP-null mutant almost lost the inhibition ability against B. cinerea growth compared to the lipopeptides from strain NCD-2 wild type. Fast protein liquid chromatography (FPLC) analysis of the lipopeptides showed that inactivation of phoR or phoP genes reduced the production of fengycin by strain NCD-2. The fengycin production abilities were compared for bacteria under low-phosphate medium (LPM) and high-phosphate medium (HPM), respectively. Results indicated that the regulation of fengycin production by the PhoR/PhoP two-component system occurred in LPM but not in HPM. Reverse transcriptional-PCR confirmed that the fengycin synthetase gene fenC was positively regulated by phoP when cultured in LPM. All of these characteristics could be partially restored by complementation of intact phoR or phoP gene in the mutant. These data indicated that the PhoR/PhoP two-component system greatly regulated fengycin production and antifungal ability in B. subtilis NCD-2 mainly under low-phosphate conditions.

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8. Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou

Journal of Integrative Agriculture 2015, 14 (5): 900-908. DOI: 10.1016/S2095-3119(14)60892-3

摘要（2269）

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A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription- polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.

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9. Perennial aneuploidy as a potential material for gene introgression between maize and Zea perennis

FU Jie, YANG Xiu-yan, CHENG Ming-jun, Lü Gui-hua, WANG Pei, WU Yuan-qi, ZHENG Ming-min, ZHOU Shu-feng, RONG Ting-zhao, TANG Qi-lin

Journal of Integrative Agriculture 2015, 14 (5): 839-846. DOI: 10.1016/S2095-3119(14)60874-1

摘要（2433）

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Hybridization, which allows for gene flow between crops, is difficult between maize and Zea perennis. In this study, we aim to initiate and study gene flow between maize and Z. perennis via a special aneuploid plant (MDT) derived from an interspecific hybrid of the two species. The chromosome constitution and morphological characters of MDT as well as certain backcross progenies were examined. Results from genomic in situ hybridization (GISH) indicate that aneuploid MDT consisted of nine maize chromosomes and 30 Z. perennis chromosomes. The backcross progenies of MDT×maize displayed significant diversity of vegetative and ear morphology; several unusual plants with specific chromosome constitution were founded in its progenies. Some special perennial progeny with several maize chromosomes were obtained by backcrossing MDT with Z. perennis, and the first whole chromosome introgression from maize to Z. perennis was detected in this study. With this novel material and method, a number of maize-tetraploid teosinte addition or substitution lines can be generated for further study, which has great significance to maize and Z. perennis genetic research, especially for promoting introgression and transferring desirable traits.

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