纳米磁性颗粒介导的辣椒遗传转化体系建立

为建立一套可操作性强、广谱性强、高效的基因编辑辣椒遗传转化体系,以辣椒B2为试验材料,磁性纳米颗粒(magnetic nanoparticles, MNPs)和载体质粒pCas9-TPC按质量比1:1复合30 min后,加入混有辣椒花粉的培养基中,于MagnetoFACTOR plate24磁板下室温放置30 min,在磁场作用下导入花粉。将携带CRISPR/Cas9基因载体的花粉进行人工授粉,果实成熟后收获种子。通过载体质粒DNA上携带的抗除草剂(草铵膦)抗性标记,检测转化情况。提取T₀代植株的DNA和RNA及T₂代DNA,以pCas9-TPC为模板设计引物进行PCR扩增,检测T₀和T₂代植株中是否存在pCas9-TPC载体,对T₀代植株进行Southern杂交检测基因编辑载体与辣椒基因组的整合情况。结果显示,平均转化效率约为63.70%,PCR检测显示T₀代植株DNA和RNA及T₂代DNA均扩增出CRISPR/Cas9载体,随机选取8株阳性植株进行Southern杂交,随机出现1~3条条带。本研究表明纳米磁性颗粒介导的花粉转化法可以在辣椒上建立高效的基因编辑转化体系,为辣椒的遗传育种提供更好的工具。     

CRISPR/Cas9介导的番茄SlMAPK6突变对植株形态的影响

促分裂原活化蛋白激酶(MAPK)级联信号参与调控植物的多种逆境、生长发育以及花青苷的合成通路。为探究SlMAPK6在番茄中的功能,本研究利用CRISPR/Cas9技术设计3个靶标位点定点编辑SlMAPK6,并获得突变体CRISPR-3和CRISPR-7。PCR和测序结果发现SlMAPK6敲除载体构建成功。通过对T₁植株进行卡那霉素基因筛选、靶位点扩增测序及行脱靶效应分析,结果发现矮番茄植株成功突变且未脱靶,与野生型对照表型相比,所有突变植株表现出根系更发达、侧枝增多的表型,表明SlMAPK6在番茄植株形态建成的过程中发挥了重要作用,为进一步探究SlMAPK6在番茄生长发育中的作用奠定了基础。     

CRISPR/Cas9定点编辑水稻LOC_Os05g31750基因位点及靶修饰位点遗传稳定性研究

为明确CRISPR/Cas9系统中Cas9蛋白的持续剪切是否会导致原已突变的靶位点在水稻代际之间传递时再次产生新的突变,本研究以水稻粳稻品种台北309为转基因受体材料,通过CRISPR/Cas9技术定点编辑了水稻基因位点LOC_Os05g31750,并获得了6株靶位点敲除成功的T_0转基因突变体植株,经三代种植收获后,进一步对携带Cas9基因的T_1和T_2群体进行靶修饰位点PCR扩增测序检测。结果表明,来源于2个T_0纯合双等位突变体T_0-8和T_0-12的所有测试后代群体的靶修饰位点序列都与对应的T_0突变体保持一致,进一步证明CRISPR/Cas9基因编辑水稻的靶修饰位点在代际之间可以稳定遗传。本研究结果为利用CRISPR/Cas9基因编辑突变体的遗传后代进行基因功能丧失后的表型分析提供了参考依据。     

紫云英与有机物料连续还田在黄泥田水稻稳产提质增效中的作用

为明确紫云英与不同有机物料还田在黄泥田改土培肥与水稻化肥减量增效中的作用，基于连续11a定位试验，研究了不施肥(T₀,CK)、单施化肥(T₁)、仅翻压紫云英(T₂)、紫云英与水稻秸秆联合还田(T₃)、紫云英与牛粪配施还田(T₄)，以及紫云英与水稻秸秆联合还田+40%化肥(T₅)处理对水稻产量、籽粒营养品质及稻田肥力的影响。结果表明，与CK相比，各施肥处理籽粒历年平均产量增幅11.4%～21.0%，秸秆平均产量增幅17.1%～40.2%，差异均显著；其中以T5提升尤为明显，其籽粒产量与秸秆产量较T1分别提高3.4%和6.6%；有效穗是产量差异的重要性状因子。各施肥处理成熟期植株地上部氮、磷、钾吸收量较CK分别增幅14.3%～30.6%、8.9%～32.7%、2.9%～47.2%，其中氮、磷吸收量以T₄最高，钾吸收量以T₅最高。与CK相比，第10年施肥处理籽粒氨基酸总量增幅11.5%～20.6%，必需氨基酸增幅11....     

连续三年施用改良剂对镉污染稻田的修复效果研究

【目的】本研究目的为探讨连续三年施用石灰、生物炭和硅肥对中度镉污染稻田的修复效果。【方法】以湖南省典型双季稻区土壤-水稻系统为研究对象,采用随机区组设计,共设对照(T₁)、施用石灰(T₂)、施用生物炭(T₃)和施用硅肥(T₄)4个处理,分析施用不同改良剂的稻田土壤理化性质和水稻镉含量特征,通过冗余分析与回归分析探究土壤因子对镉在土壤-水稻系统中迁移转运的影响。【结果】相较于T₁处理,T₂处理土壤pH显著增加至6.92±0.05,土壤有效镉降低至0.41±0.09 mg kg^-1;T₃、T₄处理与T₁处理相比,土壤pH、有效镉、活性有机质均无显著差异。T₃处理早稻和晚稻糙米镉含量最高,分别为0.54±0.07和0.11±0.04 mg kg^-1;T₂处理最低,分别为0.16±0.00和0.03±0.01 m...     

抗盐碱剂对盐碱胁迫条件下双季稻渗透调节物质及根系活力的影响

  目的  为筛选合适的抗盐碱剂供盐碱地水稻生产应用。  方法  本研究以早稻品种陵两优942和晚稻品种Y两优911为材料,采用盆栽土培法研究了几种抗盐碱剂（T₁微纳米硅、T₂矿源黄腐酸钾、T₃盐碱、T₄松土精 + 矿源黄腐酸钾）对盐碱胁迫条件下（CK₁盐碱胁迫、CK₂无盐碱胁迫）水稻渗透调节物质、根系活力及产量的影响。  结果  水稻的SPAD值、渗透调节物质、根系活力和产量会受到盐碱胁迫的显著影响,中度胁迫造成的影响高于轻度胁迫造成影响;施用抗盐碱剂有利于缓解盐碱胁迫的不利影响,且在盐碱胁迫较重时效果更明显。在中度盐碱胁迫下,与CK₁相比,施用抗盐碱剂T₁、T₂、T₃、T₄,叶片SPAD、根系活力、产量显著增加,叶片游离脯氨酸含量、可溶性糖含量显著下降;与CK₂ 相比,CK₁减产40.8%,施用抗盐碱剂则使Y两优911在盐碱胁迫条件下减产幅度显著减小,T₁、T₄、T₃、T₂分别比CK₁产量高出53.9%、31.1%、21.1%、16.3%。  结论  不同处理间综合表现以T₁（微纳米硅）和T₄处理（松土精 + 矿源黄腐酸钾）表现最佳,是盐碱地水稻增产增收的有效措施。     

含枯草芽孢杆菌肥料对大豆叶片光合作用及土壤酶活性的影响

为了减少大豆生产过程中化肥的施用量及对黑土地的保护,开展大豆田间试验,分别施用大豆专用复合肥(CK)、枯草芽孢杆菌+氨基酸型有机肥(T₁)、枯草芽孢杆菌+黄腐酸钾肥(T₂)、枯草芽孢杆菌+海藻精粉颗粒肥(T₃)、枯草芽孢杆菌秸秆腐熟有机肥(T₄)、枯草芽孢杆菌鸡粪腐熟有机肥(T_5),综合评价各种肥料对大豆光合作用和土壤酶活的作用效果。结果表明:光合参数方面,T₁、T₄和T_5处理的净光合速率较CK增加了5.0%、0.7%和8%,但差异不显著;蒸腾速率的最大值为T_5,较CK增加了30%,最小值为T₂,较CK降低了43.1%;气孔导度从高到低依次为T_5>T₄>T₁>CK>T₃>T₂,其中T₂、T₃和T_5与CK差异显著;T₂处理胞间CO_2浓度最低,较CK显著降低了12.0%,T₁、T_5处理较CK分别增加了5%和7%。叶绿素含量中,T₄和T_5的叶绿素含量均显著低于CK,T₁处理的叶绿素a含量最高,较CK增加了17.3%,T₂处理的叶绿素b含量较CK提高了2%,T₃处理与CK相比无明显变化。各处理的土壤脲酶活性均显著高于CK T_5处理为最大值,较CK提高17.7%;土壤蔗糖酶从高到低依次为T₁> T₃>CK>T₁>T₄>T₂,其中T₁较CK提高了18.9%,T₂较CK降低了27.8%;土壤碱性磷酸酶的最小值为T₃处理,酶活仅为8.55 mg·g^-1,最大值为T₄处理,较CK增加了7%。作为环境友好肥料,枯草芽孢杆菌+氨基酸型有机肥、枯草芽孢杆菌秸秆腐熟有机肥和鸡粪枯草芽孢腐熟有机肥部分代替化肥,具有应用潜力。     

隔盐方式对设施盐渍化土壤主要盐离子空间分布及酶活性的影响

针对设施土壤盐渍化日趋加重的现状,采用土柱模拟试验,以无隔盐层为对照,设置了3种隔盐层类型:砂砾层（T₁）,复合有机物料层（T₂）、砂砾+复合有机物料层（T₃）,探索不同隔盐模式的抑盐效果。结果表明:盐离子含量随着土层深度的增加而逐渐降低;就7种主要离子（Na⁺,NH₄⁺,K⁺,Ca²⁺,Cl^-,NO₃^-,SO₄^2-）总含量而言,T₃在0-10 cm,10-20 cm和20-30 cm时分别较CK降低了13.5%,0.61%和27.0%,效果较好;T₁和T₂隔盐效果不明显。0-30 cm土层,T₃分别增加了脲酶、蔗糖酶、磷酸酶活性达7.30%,4.70%,3.58%,降低了过氧化氢酶活性达8.53%;而T₁则趋势相反。说明T₃可以较好地抑制设施盐渍化土壤盐离子在耕层的聚集,同时可以提高土壤酶活性,对改善设施盐渍化土壤质量具有一定的作用。     

VIGS沉默番茄SlDCL2和SlDCL4破坏Ty-1/Ty-3对TYLCV的抗性

番茄黄化曲叶病毒(TYLCV)严重威胁茄科蔬菜作物的生产。抗性标记Ty-1和Ty-3是一对等位基因,在番茄抗TYLCV育种中应用较为广泛。为了探究Ty-1/Ty-3抗病毒分子机制,本研究以携带Ty-1/Ty-3抗性标记的番茄Y19为材料,利用病毒诱导基因沉默(VIGS)技术,沉默RNA干扰(RNAi)机制关键基因,即番茄核糖核酸内切酶编码基因2a/b/c/d(SlDCL2)和番茄核糖核酸内切酶编码基因4(SlDCL4),初步分析其在Ty-1/Ty-3抗TYLCV中的功能。PCR与测序结果发现SlDCL2、SlDCL4的VIGS沉默载体pTRV2∶SlDCL2和pTRV2∶SlDCL4构建成功。以沉默番茄八氢番茄红素脱氢酶(SlPDS)基因为标记植株,定量PCR检测SlDCL2、SlDCL4沉默株系中SlDCL2、SlDCL4基因的沉默效率,结果显示SlDCL2、SlDCL4沉默株系中对应基因的表达均小于对照植株的50%,表明SlDCL2、SlDCL4沉默载体确实可以降低对应基因的表达,且不影响其他DCL基因的表达。VIGS沉默植株SlDCL2、SlDCL4基因后接种TYLCV,接种TYLCV 30dpi结果显示,Y9材料表现出明显的TYLCV病毒症状,通过比较植株发病严重度发现,SlDCL2、SlDCL4沉默株系发病严重度分别为1.97、2.35,显著高于空载体注射株系(0.14)和对照株系(0.07)。本研究结果表明RNAi通路关键基因SlDCL2、SlDCL4在Ty-1/Ty-3抗TYLCV中发挥着重要作用,这为利用Ty-1/3抗性标记基因育种奠定了基础。     

水稻淡黄叶突变体xws的光合特性与基因定位

为了解析水稻叶绿体发育和高光效育种中光合作用的机理,本研究以不育系P88S群体中发现的一株淡黄叶自然突变体xws为试验材料,对该突变体进行表型鉴定和光合特征分析,并对其进行精细定位和候选基因分析。结果表明,xws在整个生育期叶片、茎和穗均呈淡黄色。与野生型相比,xws叶片、茎和穗的叶绿素a、叶绿素b和类胡萝卜素含量均显著下降。透射电镜观察结果发现,与野生型相比,xws嗜锇小体消失,类囊体数量减少。xws的净光合效率、气孔导度、蒸腾速率、胞间CO₂浓度和叶绿素荧光参数与野生型相比均降低。以xws与R032杂交的F₂群体作为定位群体,将基因精细定位至水稻第3号染色体分子标记WY242和WY119之间,其物理距离为51.7 kb。本研究结果为进一步研究水稻叶绿体发育,从而提高水稻光合作用和分子调控网络途径提供了理论支撑。     

Soil physical responses to novel rice cultural practices in the rice–wheat system: Comparative evidence from a swelling soil in Nepal

Soil puddling in advance of rice (Oryza sativa L.) transplanting disperses surface aggregates and generates compaction at depth. As a management scheme for rice, puddling is typically considered advantageous for maximizing resource availability and yield. However, some experimental findings suggest a conflict between edaphic conditions created by this establishment technique and the performance of subsequent non-rice crops like wheat (Triticum aestivum L.). At a site in the mid-hills region of Nepal on a silt loam soil with vertic characteristics, we compared the impact of six rice tillage (surface tillage—T₁, shank subsoiler—T₂, shank subsoiler + moldboard plough—T₃) and establishment (soil puddling + transplanting—TPR, direct seeding—DSR) combinations on soil physical properties over two cycles of the rice–wheat rotation. For the rice season, 0–20 cm saturated hydraulic conductivity (K_sat) in the DSR plots was 2.6 and 4.3 times higher than their TPR counterparts in the first (Y₁) and second (Y₂) years, respectively (TPR-Y₁ = 93 mm day⁻¹, DSR-Y₁ = 241 mm day⁻¹, TPR-Y₂ = 133 mm day⁻¹, DSR-Y₂ = 582 mm day⁻¹), whereas tillage method did not significantly influence K_sat in this soil layer. The impact of rice establishment method was reflected in higher TPR bulk densities in the 5–10 (DSR = 1.19 g cm⁻³, TPR = 1.24 g cm⁻³) and 10–15 cm (DSR = 1.24 g cm⁻³, TPR = 1.29 g cm⁻³) depth increments in the wet season. Although none of the treatments significantly influenced the position or thickness of the plough sole, penetration resistance profiles suggest that vertical fractures with reduced soil strength were created within the pan region by deep tillage (T₂ and T₃), although these features were not associated with higher hydraulic conductivities from 20 to 50 cm. As the soils dried at the end of the rice season, crack propagation in the deep tilled plots (T₂ and T₃) was more pervasive. During the wheat season, comparable bulk density profiles and soil moisture retention characteristics across the treatments suggest that many of the edaphic changes induced by contrasting rice tillage and establishment practices did not persist in the self-mulching, vertic soils at our site. Conversely, significant increases in K_sat among the DSR plots from Y₁ to Y₂ (Y₁ = 241 mm day⁻¹, Y₂ = 582 mm day⁻¹) imply a temporal element to soil structural regeneration with adoption of direct seeding.     

经济作物替代薯稻三熟制早稻对肥料利用、土壤养分和经济效益的影响

为缩短“薯-稻-稻”三熟制作物早稻的生育期,促进“薯-稻-稻”茬口顺畅衔接,提高整体经济效益,本研究以不同经济作物替代早稻,分别在2016年惠东县和2017年白云区试点进行田间试验,共设置5个处理:休耕T₀(空白对照,不种任何作物)、常规处理T_CK(早稻)、T₁(黄瓜)、T₂(豆角)、T₃(甜玉米),探究不同春播经济作物生育期、产量、种植效益和肥料效率及土壤酶活的差异。结果表明,种植黄瓜、豆角和甜玉米替代早稻均可明显增加经济效益和缩短生育期。其中,T₃效果最优,与T_CK相比,单位面积种植效益增加了1.32~3.72 元·m^-2,生育期缩短了37~43 d,收获指数提高了18.6%~20.1%;同时,增加了土壤有效磷和速效钾含量,增强了土壤脲酶、磷酸酶和过氧化氢酶的活性。综上,甜玉米等经济作物代替早稻形成的“经济作物-中晚稻-冬种马铃薯”三熟轮作模式可以使作物茬口衔接更加顺畅,提升三季作物的整体经济效益,对农民增收具有积极作用。本研究为优化广东乃至华南“薯-稻-稻”三熟区的种植模式提供了理论基础和实践依据。     

Efficiency and energy use in puddling of lowland rice grown on Vertisols in Central India

Transplanting of rice seedling in puddled soil is one of the most widely used cultivation practices. The present research is aimed at determining what specific implements are needed to obtain optimal puddle bed for transplantating. Puddling experiments were carried out by the use of pair of bullocks with traditional country plough (T₁), pair of bullocks with lug wheel puddler (T₂), power tiller with rotary puddler (T₃), tractor with cage wheel and 9-tine cultivator (T₄) and tractor with cage wheel and rotavator (T₅). One summer ploughing was done at friable moisture condition (18.6% db) and then tilled soil was flooded to saturation (24 h) for preparation of puddled bed. Weeding efficiency, puddling depth, percentage increase in bulk density, puddling index, percolation rate and grain yield of paddy were studied for the above treatments. Puddling performance by different implements in comparison to the traditional animal drawn country plough (T₁) shows that there is a definite reduction in time requirement for field preparation. Increase in weeding efficiency, bulk density, grain yield and puddling index were also observed. The highest values of weeding efficiency and puddling index were found 98.6% and 79.3, respectively, for rotavator (T₅). The total time requirement for preparation of puddle field for treatment T₄ (tractor with cultivator) was found to be the lowest (9.4 h ha⁻¹) with 67% weeding efficiency and 62.7 puddling index as compared to all other alternatives tested. Energy requirement for preparation of puddle field was found highest (2390 MJ ha⁻¹) for rotavator (T₅) followed by T₃, T₄, T₁, and T₂ treatments.     

Rice roots and CH4 oxidation: the activity of bacteria, their distribution and the microenvironment

Irrigated rice fields account for 10–30% of global methane emissions. Rice plants ventilate the soil and enlarge the oxic–anoxic interface by their root system, thus supplying the necessary O₂ to aerobic CH₄ oxidizing bacteria (MOB). Rice plants (Oryza sativa type japonica var. Roma) were grown in microcosms in a greenhouse. The roots were sandwiched between two blocks of flooded rice field soil separated by a nylon gauze bag. A root mat developed which mimicked the dense root texture in the upper layer of a natural rice field. Flux measurements under oxic and anoxic conditions showed that CH₄ was oxidized with a constant rate of 19% of the anoxically emitted CH₄, suggesting that CH₄ oxidation in the rhizosphere was at least sometimes limited by CH₄ availability. Washed rice roots could both produce and oxidize CH₄, depending upon incubation conditions. CH₄ production by washed rice roots accounted for at most 10% of the CH₄ emitted under anoxic conditions. Initial CH₄ oxidation rates of washed roots equaled oxidation rates calculated from the difference between oxic and anoxic fluxes in situ. Oxidation rates became twice as high after an induction period of 20 h, indicating a limitation by O₂ or CH₄ in situ. The micro-environmental conditions near to the root mat were measured using microelectrodes for O₂, redox potential and NH₄⁺ and diffusion probes for CH₄. Up to 42 μM O₂ was detected in the root mat and concentrations were >2.5 μM in 45% of all measurements. In the bulk soil, no O₂ was detected below 2 mm depth, but the root mat significantly increased the redox potential. Plant roots and associated bacteria decreased porewater CH₄ and NH₄⁺ concentrations. In the root mat, concentrations of dissolved CH₄ were below the detection limit of our probes (<5 μM). Cell numbers of MOB increased with time in the rhizosphere and in the rhizoplane. MOB and aerobic heterotrophic bacteria (AHB) each numbered from 10⁶ to 10⁸ cells g⁻¹ dry weight of soil or root biomass). Active MOB occurred near to a root mat similar to the dense root texture in the upper layer of rice fields. We speculate about O₂ or CH₄ limitation of MOB.     

基于宽窄行种植模式下稻秸非均匀性覆盖对土壤特性及小麦产量的影响

为探究宽窄行种植模式下稻秸非均匀性覆盖还田对土壤理化特性和小麦产量的影响,在大田条件下,本研究通过前期不同行间距配置试验筛选出30 cm+15 cm的优势宽窄行组合,并在此基础上设置5个不同梯度的宽窄行稻秸分布比例处理(T₁:0、T₂:25%、T₃:50%、T₄:75%、T₅:100%),分析窄行(苗带)土壤理化性质和小麦产量变化情况。结果表明,0~10 cm为土壤温度和含水率变化敏感层。随着窄行稻秸覆盖量的升高,小麦生育前期土壤增温幅度增大,中后期降温效果更明显,土壤保墒性能增强,拔节期各处理间土壤含水率差异增大(增加1.2%~3.4%)。窄行稻秸覆盖量增加可在一定程度上降低土壤容重,提高土壤孔隙度,这种增减效应与覆盖量呈一定正比关系,但与生育时期无关;开花期和成熟期,土壤有机质、全氮、速效磷和速效钾含量均随窄行稻秸覆盖量增加呈先升后降趋势,总体上表现出适量稻秸覆盖(T₃:窄行秸秆覆盖量为均匀覆盖时窄行秸秆量的1/2)更有利于增加土壤养分含量。小麦产量及其构成因素均随窄行稻秸覆盖比例的不断增加呈降低趋势,其中有效穗数和产量(降幅为4.0%~31.7%)均显著降低(P<0.05)。可见,在稻秸全(大)量非均匀还田和晚播情形下,只有合理配比宽窄行秸秆覆盖量,并适当增加小麦播种量,保证足够基本苗,才能达到稳产肥地的协同效应。     

硝态氮促进水稻生长和氮素吸收的生理机制

Rice is being increasingly cultivated in intermittently irrigated regious and also in aerobic soil in which Nitrate (NO₃^-) plays important role in nutrition of plant. However, there is no information regarding the influence of nitrate on the overall growth and uptake of nitrogen (N) in rice plant. Solution culture experiments were carried out to study the effects of NO₃^- on the plant growth, uptake of N, and uptake kinetics of NH₄⁺ in four typical rice (Oryza sativa L.) cultivars (conveutioual indica, conventional japonica, hybrid indica, and hybrid japonica), and on plasma membrane potential in roots of two conventional rice cultivars (indica and japonica) at the seedling stage. The results obtained indicated that a ratio of 50/50 NH₄⁺-N/NO₃^--N increased the average biomass of rice shoots and roots by 20% when compared with that of 100/0 NH₄⁺-N/NO₃^--N. In case of the 50/50 ratio, as compared with the 100/0 ratio, total N accumulated in shoots and roots of rice increased on an average by 42% and 57%, respectively. Conventional indica responds to NO₃^- more than any other cultivars that were tested. The NO₃^- supply increased the maximum uptake rate (V_max) of NH₄⁺ by rice but did not show any effect on the apparent Michaelis-Menten constant (K_m) value, with the average value of V_max for NH₄⁺ among the four cultivars being increased by 31.5% in comparison with those in the absence of NO₃^-. This suggested that NO₃^- significantly increased the numbers of the ammonium transporters. However, the lack of effect on the K_m value also suggested that the presence of NO₃^- had no effect on the affinity of the transporters for NH₄⁺. The plasma membrane potential in the roots of conventional indica and japonica were greatly increased by the addition of NO₃^-, suggesting that NO₃^- could improve the uptake of N by roots of the rice plant. In conclusion, the mechanisms by which NO₃^- enhances the growth and N uptake of rice plant was found by the increased value of V_max of NH₄⁺ and increased plasma membrane potential. Thus promotion of nitrification in paddy soil is of great significance for improving the production of rice.