水稻CSC11介导干热风/干旱诱导的钙信号调控雄蕊发育

【目的】水稻花期偶遇干热风/干旱,导致脆弱的生殖细胞快速失水,极大地降低产量,这一过程中钙离子作为通用的第二信使传导了干旱或其他逆境信号,但背后的分子机制尚不清楚。分析钙离子透过性胁迫反应阳离子通道家族（calcium-permeable stress-responsive cation channels,CSCs）基因的生理和分子功能,为研究作物干热风的感应机制提供新的理论基础和思路。【方法】采用电生理学和遗传学方法,利用双电极电压钳技术在水稻中鉴定得到一个具有典型特征的受体类-钙通道蛋白,名为OsCSC11,对其蛋白序列进行生物信息学和进化关系分析。运用qRT-PCR和GUS报告基因活性分析确认OsCSC11的表达模式,在拟南芥原生质体细胞和洋葱表皮细胞中瞬时表达OsCSC11-GFP融合蛋白,验证OsCSC11的亚细胞定位;同时利用CRISPR/Cas9基因编辑技术获得OsCSC11的突变体,并通过细胞学等手段分析突变体表型和相关生理功能。【结果】蛋白序列比对发现,OsCSC11具有CSCs家族成员典型的保守结构域DUF221,但与其他成员序列差异大,存在不同于其他成员的特异结构域（motif）属于独立的亚家族。OsCSC11主要在水稻的花药和叶片中表达,进一步分析发现全长OsCSC11处于静息状态,可被高渗透溶液激活;但是删除N端156氨基酸（TM1-3）之后的OsCSC11^ΔTM1-3具有组成型的通道活性,特异选择钙、镁二价阳离子;推测TM1-3是这类通道的受体结构域,感应干热风胁迫,而OsCSC11^ΔTM1-3区域负责钙信号产生。OsCSC11和OsCSC11^ΔTM1-3均定位在细胞质膜上,与其干热风的受体功能相适应。与野生型相比,功能缺失突变体oscsc11-1和oscsc11-2的雄蕊较小、花药表面蹙皱,整体多呈弯曲状态,花粉含水量较低,败育率高达60%—70%。【结论】OsCSC11是水稻感应短期干热风/干旱刺激、介导钙离子内流,调控花药水分状态和花粉发育的受体类钙通道,可能参与了水稻雄蕊应对干热风的原初感应过程。     

基于叶片反射光谱估测水稻氮营养指数

【目的】基于叶片反射光谱建立快速、无损监测水稻氮营养指数（nitrogen nutrition index,NNI）的估算模型。【方法】2018—2019年开展2个水稻品种（徽两优898和Y两优900）及5个氮肥梯度（施氮量为0、75、150、225和300 kg·hm^-2,分别记为N₀、N₁、N₂、N₃、N₄）的田间小区试验,测定关键生育期不同叶位叶片反射光谱和植株NNI,构建多种光谱指数的水稻NNI监测模型。【结果】单叶及叶位组合的敏感波段均分布在540 nm的绿光波长处,其与近红外波段构成的窄波段比值指数SR（R₉₀₀,R₅₄₀）可较好反演水稻NNI。但不同叶位叶片窄波段比值指数与水稻NNI的预测精度表现不同,顶3叶（L₃）预测精度最好（R²=0.731,RMSE =0.130,RE=11.6%）,顶2叶（L₂）次之（R²=0.707,RMSE =0.136,RE =12.2%）,顶1叶（L₁）最差（R²=0.443,RMSE =0.187,RE =14.7%）;顶2叶和顶3叶组合平均光谱（L₂₃）的预测精度优于单叶水平和其他叶位组合（R²=0.740,RMSE =0.128,RE =11.5%）。再将窄波段比值指数SR（R₉₀₀,R₅₄₀）近红外与绿光区域分别重采样50 nm和10 nm,所构建的宽波段比值指数SR[AR_{（900±50）},AR_{（540±10）}]模型精度较SR（R₉₀₀,R₅₄₀）未明显降低,且在L₂₃水平下2个模型的模型精度和预测精度基本一致（R²=0.740,RMSE =0.128,RE =11.5%）。水稻NNI小于1时与产量呈线性的正相关关系（P<0.05）,大于1时产量趋于平稳。【结论】L₂和L₃叶片反射光谱为监测水稻NNI的敏感叶位,其中叶位组合L₂₃可提高模型预测精度。基于叶片反射光谱构建的多种波段比值指数（SR（R₉₀₀,R₅₄₀）和SR[AR_{（900±50）},AR_{（540±10）}]）可快速估测水稻NNI,从而为不同传感器对水稻氮营养指数估测监测研究提供了理论依据。     

小麦种质资源成株期氮效率评价及筛选

【目的】建立小麦成株期氮效率评价方法,挖掘和筛选氮高效种质资源,为小麦氮效率的生理机制研究和氮高效品种的选育提供理论依据和材料基础。【方法】2018—2020年,以108份不同基因型小麦品种为材料,采用大田试验,设置4个氮肥处理水平（0、180、240和360 kg·hm^-2）,调查不同氮水平下小麦株高、穗长、旗叶长、旗叶宽、茎粗、可育小穗数、穗粒数、千粒重、粒长、粒宽和单穗产量11个农艺及产量性状,利用模糊隶属函数法、主成分分析法以及聚类分析法对小麦品种进行耐氮性评价和基因型差异分类。【结果】连续2年的数据结果显示,在低氮胁迫下,株高、穗长、旗叶长、旗叶宽、茎粗、可育小穗数、穗粒数和单穗产量8个性状均受到不同程度的抑制,其中,旗叶长对氮胁迫的敏感程度最大。主成分分析提取4个主成分,贡献率分别为39.766%、16.661%、9.361%和9.275%,累积贡献率达75.064%。以耐低氮性综合评价D值进行聚类分析,将供试小麦品种划分为强耐低氮型、耐低氮型、中间型、较敏感型和敏感型5类。筛选出耐低氮型小麦品种5份,温麦19、西农529、石4185、陇麦212和丰抗2,强耐低氮型小麦品种2份,中麦875和西农158。与低氮胁迫不同,高氮胁迫仅抑制茎粗、千粒重、粒长、粒宽和单穗产量5个性状,株高、穗长、旗叶长、旗叶宽、可育小穗数和穗粒数6个性状值随施氮量上升而增加。4个主成分的贡献率分别为31.348%、20.387%、12.452%和9.850%,累积贡献率达74.037%。依据耐高氮性综合评价D值,将供试小麦品种划分为耐高氮型、中间型、高氮较敏感型和高氮敏感型4类。鉴定出耐高氮型小麦品种9份,包括兰考矮早8、良星99、农大179、豫农9901、兰考926和郑农46等。基于籽粒产量和氮综合评价D值,将108份小麦品种划分为4种氮效率类型,双高效型（西农158和陇麦212等）、低氮高效型（西农585和石4185等）、高氮高效型（长丰1号和中种麦10号等）和双低效型（金丰7183和泛麦5号等）。【结论】供氮水平对小麦产量相关性状指标有显著影响,基于小麦种质间氮吸收与利用效率的差异,结合3种分析方法,可以准确评价小麦种质资源成株期氮效率状况。     

基于新遗传连锁图谱的豇豆抗豆象QTL定位

【目的】豆象是危害豇豆最主要的仓储害虫。发掘豇豆抗豆象基因为抗性品种的选育,以及减少豆象对豇豆生产的危害奠定基础。【方法】以中豇1号（感）和Pant-lobia-1（抗）为亲本构建的包含282个株系的RIL群体为研究材料,利用人工接种法分别对282个株系接种绿豆象和四纹豆象,进行抗豆象表型鉴定,并利用两亲本对3 992个来源于绿豆、小豆和豇豆的SSR标记进行多态性筛选,然后利用筛选到的多态性标记对282个株系进行基因型分析,最后结合RIL群体各株系抗豆象表型鉴定数据和基因型分型数据,采用完备区间作图法（ICIM-ADD）进行抗豆象QTL定位分析,在此基础上构建遗传连锁图谱,并定位豇豆抗豆象基因。【结果】中豇1号和F₁籽粒的被害率均为100%,Pant-lobia-1籽粒的被害率分别为22.5%和42.5%。推测Pant-lobia-1对绿豆象和四纹豆象的抗性均为隐性遗传;筛选到182个多态性标记,利用这些多态性标记构建了一个包含11个连锁群的豇豆遗传连锁图谱,图谱总长1 065.23 cM,相邻标记间平均遗传距离为5.85 cM;经2种豆象处理,分别在连锁群1和连锁群5上检测到2个稳定的QTL位点,暂定名为vubr1-1和vubr5-1,其中vubr1-1位于标记XD11-44和HAAS_VR_2274之间,标记间的遗传距离为7.6 cM,在2种豆象处理中分别可以解释表型变异的7.16%和6.92%;vubr5-1位于标记XD1-14和CP185之间,标记间的遗传距离为2.90 cM,在2种豆象处理中分别可以解释表型变异的6.96%和6.37%。【结论】构建了一个包含11个连锁群、182个多态性标记的豇豆遗传连锁图谱,检测到2个与抗豆象相关的QTL位点。     

不同提取方法对红松籽油提取效果及功能性质的影响

  目的  立足于溶剂法,通过对比超声波、微波、光波单独处理较短时间的红松籽油的得率,以及处理后单独浸提红松籽油得率的增加情况,明确在红松籽油提取过程中处于主导作用的方法,同时确认4种提取方法对红松籽油的理化指标、脂肪酸和抗氧化能力的影响。  方法  按照GB/T 5009.229—2016《食品中酸价的测定》、GB/T 5532—2008《动植物油脂 碘值的测定》和GB/T 5009.227—2016《食品中过氧化值的测定》分别测定4种方法提取的红松籽油的酸值、碘值和过氧化物值,采用气相色谱–质谱联用法测定红松籽油中脂肪酸的种类和质量分数,采用分光光度法测定红松籽油对DPPH·和ABTS+·的清除能力。  结果  正己烷作为最佳提取溶剂,当提取温度25 ℃,液料比18 mL/g,浸提时间5 h红松籽油得率为65.52%。超声波法（300 W,10 min）、微波法（380 W,100 s）、光波法（400 W,11 min）单独处理时的红松籽油得率分别为67.63%、62.26%、58.25%。上述3种方法处理后再单独使用正己烷浸提,使总提取时间达到5 h后的红松籽油得率分别增加了2.18%、4.50%、6.10%。4种方法提取的红松籽油符合GB/T 2716—2018《食品安全国家标准 植物油》中酸值（≤ 4 mg/g）、过氧化值（≤ 0.25 g/100 g）的限量标准。溶剂法制备出的红松籽油的酸值和过氧化值均最低（P < 0.05）。脂肪酸质量分数由高到低分别是亚油酸（44.54% ~ 46.32%）、油酸（28.29 % ~ 28.83%）和皮诺敛酸（13.15% ~ 14.51%）。溶剂法、超声波法、微波法、光波法所提取的红松籽油清除DPPH·和ABTS+·的IC₅₀值分别是9.41、8.80、9.43、9.61 g/L和5.10、5.51、6.10、5.43 g/L。  结论  对超声波法、微波法和光波法单独作用及浸提后得率进行比较,发现超声波法、微波法和光波法在提取过程中起主导作用。与溶剂法、微波法和光波法相比,超声波法提取的红松籽油得率高,不饱和脂肪酸质量分数高,抗氧化能力强,能够达到较好的提取效果,此方法应用于红松籽制油工业中具有一定优势。      

Expression profiling of transgenes (Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the Cry1Ac gene has revolutionized insect pest control since its adoption, although the development of resistance in insect pests has reduced its efficacy.  After 10 years of cultivating Bacillus thuringiensis (Bt) cotton with a single Cry1Ac gene, growers are on the verge of adopting Bt cotton that carries the double gene (Cry1Ac+Cry2A) due to its better effectiveness against insect pests.  Thus, the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.  The expression levels of the Cry1Ac and Cry2A genes were evaluated in the leaves of 10 genotypes (2 parents and 8 F₁ hybrids) at 30 days after sowing (DAS), while samples of leaves, bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.  The F₁ hybrids were developed through reciprocal crosses between two Bt (CKC-1, CKC-2) and two non-Bt (MNH-786, FH-942) parents.  The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay (ELISA).  The results showed that the MNH786×CKC-1 hybrid had the highest concentrations of Cry1Ac gene at 30 DAS (3.08 µg g^–1) and 110 DAS (1.01 µg g^–1) in leaves.  In contrast, the CKC-2×MNH-786 hybrid showed the lowest concentrations of Cry1Ac gene at 30 DAS (2.30 µg g^–1) and 110 DAS (0.86 µg g^–1).  The F₁ hybrid FH-942×CKC-2 showed the highest concentrations of Cry2A gene at 30 DAS (8.39 µg g^–1) and 110 DAS (7.74 µg g^–1) in leaves, while the CKC-1×MNH-786 hybrid expressed the lowest concentrations of Cry2A gene at 30 DAS (7.10 µg g^–1) and 110 DAS (8.31 µg g^–1).  A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS, whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.  When the expression pattern was compared between various plant parts in genotype CKC-2, it was found that leaves had higher concentrations of Cry1Ac (3.12 µg g^–1) and Cry2A (8.31 µg g^–1) at 70 DAS, followed by bolls (Cry1Ac (1.66 µg g^–1) and Cry2A (8.15 µg g^–1)) and flowers (Cry1Ac (1.07 µg g^–1) and Cry2A (7.99 µg g^–1)).  The genotype CKC-2 had higher concentrations of Cry1Ac (3.12 µg g^–1) and Cry2A (8.31 µg g^–1) in the upper canopy but less accumulation (2.66 µg g^–1 of Cry1Ac, 8.09 µg g^–1 of Cry2A) in the lower canopy at 70 DAS.  Similarly, at 110 DAS, the expression levels of Cry1Ac and Cry2A in upper and lower canopy leaves were 1.52 and 7.92 µg g^–1, and 0.99 and 7.54 µg g^–1, respectively.  Hence, the current study demonstrates that different genotypes showed variable expression for both of the Cry1Ac and Cry2A genes during plant growth due to different genetic backgrounds.  The Cry2A gene had three-fold higher expression than Cry1Ac with significant differences in expression in different plant parts.  The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1Ac and Cry2A for sustainable cotton production worldwide.     

Predictive models of drought tolerance indices based on physiological,morphological and biochemical markers for the selection of cotton (Gossypium hirsutum L.) varieties

The use of tolerant crop varieties is a strategy that mitigates the water deficit effect in a sustainable way. The generation of these varieties is more efficient when variables associated with this tolerance have been identified, since they can facilitate the breeding processes. This study aimed to establish the relationships between water deficit tolerance of four cotton varieties (Nevada-123, Oasis-129, Guatapuri, and Festivalle) and morphological variables (monopodial branches, boll weight, root/shoot ratio, and leaf and root dry matter), physiological variables (relative water content, net photosynthesis, stomatal conductance, electron transport rate, photochemical quenching, photochemical efficiency of PSII, chlorophyll a/b ratio (Chl a/b), C¹²/C¹³ isotope ratio, and electrolyte leakage), and biochemical variables (contents of sugars, proline, carotenoids, and malondialdehyde). Furthermore, calibrated predictive models of the drought tolerance indices were developed based on the key variables identified. For this purpose, a pot experiment was established where plants were subjected to a moderate or severe water deficit during the blooming stage for 12 days. The stress tolerance index (STI) and mean productivity (MP) were calculated. For the evaluated variables, the differences between well-watered and water deficit plants (Δ) were calculated and ANOVA, partial least squares, Pearson's correlation, and multiple linear regression analyzes were performed. A model was generated that explained 95% of the STI and was composed of Δmalondialdehyde, Δproline, and Δboll weight. For MP, the model was comprised of Δstomatal conductance, Δroot/shoot ratio, and ΔChl a/b, and explained 89% of the MP. The analysis of the assessed variables allowed the identification of key variables and the development of calibrated predictive models that can be used in screening to obtain cotton varieties with different levels of water deficit tolerance.     

Tomato mottle mosaic virus: Characterization,resistance gene effectiveness,and quintuplex RT-PCR detection system

Tomato mottle mosaic virus (ToMMV), an economically important species of the genus Tobamovirus, causes significant loss in yield and quality of tomato fruits. Here, we identified the Shandong isolate of ToMMV (ToMMV-SD) collected from symptomatic tomato fruits in Weifang, Shandong Province of China. ToMMV-SD caused symptoms such as severe mosaic, mottling, and necrosis of tomato leaves, yellow spot and necrotic lesions on tomato fruits. The obtained full genome of ToMMV-SD was 6 399 nucleotides (accession number MW373515) and had the highest identity of 99.5% with that of isolate SC13-051 from the United States of America at the genomic level. The infectious clone of ToMMV-SD was constructed and induced clear mosaic and necrotic symptoms onto Nicotiana benthamiana leaves. Several commercial tomato cultivars, harboring Tm-2² resistance gene, and pepper cultivars, containing L resistance gene, were susceptible to ToMMV-SD. Plants of Solanum melongena (eggplant) and Brassica pekinensis (napa cabbage) showed mottling symptoms, while N. tabacum cv. Zhongyan 100 displayed latent infection. ToMMV-SD did not infect plants of N. tabacum cv. Xanthi NN, Brassica rapa ssp. chinensis (bok choy), Raphanus sativus (radish), Vigna unguiculata cv. Yuanzhong 28-2 (cowpea), or Tm-2² transgenic N. benthamiana. A quintuplex RT-PCR system differentiated ToMMV from tomato mosaic virus, tomato brown rugose fruit virus, tobacco mosaic virus, and tomato spotted wilt virus, with the threshold amount of 0.02 pg. These results highlight the threat posed by ToMMV to tomato and pepper cultivation and offer an efficient detection system for the simultaneous detection of four tobamoviruses and tomato spotted wilt virus infecting tomato plants in the field.     

Heterosis and heterotic patterns of maize germplasm revealed by a multiple-hybrid population under well-watered and drought-stressed conditions

Understanding the heterosis in multiple environments between different heterotic groups is of fundamental importance in successful maize breeding. A total of 737 hybrids derived from 41 maize inbreds were evaluated over two years, with the aim of assessing the genetic diversity and their performance between heterotic groups under drought-stressed (DS) and well-watered (WW) treatments. A total of 38 737 SNPs were employed to assess the genetic diversity. The genetic distance (GD) between the parents ranged from 0.05 to 0.74, and the 41 inbreds were classified into five heterotic groups. According to the hybrid performance (high yield and early maturity between heterotic groups), the heterosis and heterotic patterns of Iowa Stiff Stalk Synthetic (BSSS)×Non-Stiff Stalk (NSS), NSS×Sipingtou (SPT) and BSSS×SPT were identified to be useful options in China's maize breeding. The relative importance of general and specific combining abilities (GCA and SCA) suggests the importance of the additive genetic effects for grain yield traits under the WW treatment, but the non-additive effects under the DS treatment. At least one of the parental lines with drought tolerance and a high GCA effect would be required to achieve the ideal hybrid performance under drought conditions. GD showed a positive correlation with yield and yield heterosis in within-group hybrids over a certain range of GD. The present investigation suggests that the heterosis is due to the combined accumulation of superior genes/alleles in parents and the optimal genetic distance between parents, and that yield heterosis under DS treatment was mainly determined by the non-additive effects.     

5%调环酸钙EA对小麦抗倒伏和产量及其相关因素的影响

为了探讨调环酸钙对小麦产量相关因素及安全性的影响,以小麦品种‘豫麦49’为材料,返青后施用不同剂量的5%调环酸钙EA开展田间试验。结果表明,小麦返青期施用5%调环酸钙EA 150~ 600 g/hm²,可矮化植株高度,降低基部节间长度、增加茎粗和茎壁厚度。调环酸钙能提高小麦穗数、穗粒数、千粒重和产量。返青期施用5%调环酸钙EA 300 g/hm²和5%调环酸钙EA 300 g/hm²+0.01%芸苔素内酯SL 150 mL/hm²的产量较高,分别达7410.0 kg/hm²和7560.0 kg/hm²,较对照(CK)分别增产10.7%和13.0%。5%调环酸钙EA对小麦安全无药害,建议在小麦上推广使用。