利用CRISPR/Cas9系统编辑日本结缕草ZjSGR基因技术研究

日本结缕草(Zoysia japonica)是一种常见的具有众多优良性状的暖季型草种,秋冬季节在我国北方地区会较早出现叶片脱绿现象,在一定程度上限制了日本结缕草在北方的大面积使用。ZjSGR是从结缕草中分离出来的一种衰老诱导基因,负调控叶片滞绿性状。本研究利用CRISPR/Cas9系统,通过基因枪介导的遗传转化方法实现对日本结缕草ZjSGR基因的编辑,经测序验证获得1株单碱基缺失突变植株。突变植株叶片深绿,在正常生长温度(28~30℃)条件下,和对照相比,叶绿素含量更高(P<0.05)。本研究为进一步阐述ZjSGR基因在日本结缕草滞绿性状调控中的作用机理提供了基础研究材料,也为培育日本结缕草滞绿品种育种工作奠定了基础。     

The mechanical roles of the clasping leaf sheath in cereals: Two case studies from oat and wheat plants

Stem lodging is a common problem in cereal crop production and a main constraint for grain yield improvement. The leaf sheath that surrounds and protects the hollow internodes of stem could provide the plants with a great physical support. However, this biomechanical function has been ignored for several decades in cereal crops. This study aimed to examine the biomechanical properties of basal stem internodes and lodging susceptibility of the whole plants with or without the clasping leaf sheath in wheat (Triticum aestivum L.) and oat (Avena sativa L.) among different genotypes and agronomic practices (including planting densities and nitrogen application rates). The main objective was to quantify the mechanical role of the leaf sheath in oat and wheat crops by a “safety factor” method. On average, the leaf sheath contributed 40%, 68% and 38% of the overall stem bending strength, flexural rigidity and safety factor, in oat, while it accounted for 11%, 24% and 10%, respectively, in wheat plants. The significant contribution of the leaf sheath is due to its vital role in enlarging the peripheral position (i.e., second moment of area) and stiffness (i.e., Young's modulus). The contribution ratios (%) were found to be higher in oat than in wheat plants, due to the greater mass density of leaf sheath and more proficient/prevailing stay-green capability in oat genotypes. This study emphasizes the important mechanical role of clasping leaf sheath on stem internodes of cereals and indicates that the stay-green trait of the leaf sheath can be exploited to design appropriate varieties with improved lodging resistance and great yield potential.     

外源6-BA和不同用量氮肥配合对小麦花后叶片功能与荧光特性的调控效应

【目的】探讨外源细胞分裂素(6-BA)和不同用量氮肥对小麦花后光合特性的调控效应,为激素与氮肥配合施用提高小麦光合生产力提供理论依据。【方法】试验选用持绿型品种汶农6号和非持绿型品种济麦20,设置N0(0)、N1(240 kg·hm-2)、N2(360 kg·hm-2)3个氮肥用量,同时,花后连续3 d叶面喷施25 mg·L-1 6-苄基腺嘌呤(6-BA)及300 mg·L-1洛伐他汀(Lovastatin),用量100 m L·m-2。开花后每隔7 d取旗叶,测定叶绿素含量、MDA含量、抗氧化酶活性等生理指标,用高效液相色谱法测定4种内源激素含量,利用脉冲调制式荧光仪测定不同处理下旗叶叶绿素荧光诱导的动力学参数。【结果】喷施外源6-BA显著提高两品种小麦旗叶花后不同时期最大光化学效率(Fv/Fm)、实际光化学效率(ΦPSII)、光合电子传递速率(ETR)以及光化学猝灭系数(q P),而喷施外源洛伐他汀对上述指标产生显著降低作用。喷施外源6-BA使N0、N1、N2处理下济麦20旗叶ΦPSII分别提高12.08%、14.21%、9.43%,汶农6号旗叶ΦPSII分别提高12.44%、14.84%、11.58%;喷施外源6-BA使N0、N1、N2处理下济麦20旗叶ETR分别提高16.57%、25.81%、18.83%,汶农6号旗叶ETR分别提高13.88%、23.58%、22.80%。两品种其他荧光参数指标表现出以下规律,即6-BA与N1配合对小麦旗叶Fv/Fm、ΦPSII、ETR以及q P的提高效应均高于单一喷施6-BA或6-BA与N2配合。同时,品种、氮肥、激素单一效应及激素与氮肥配合对ΦPSII、ETR、q P影响显著,品种、激素单一效应对Fv/Fm影响显著,而激素与不同用量氮肥配合对Fv/Fm无显著影响。叶面喷施细胞分裂素抑制剂洛伐他汀使N0、N1、N2处理下济麦20旗叶ETR分别降低22.71%、12.06%、11.92%,两品种其他荧光参数指标Fv/Fm、ΦPSII、q P均表现出下降趋势,而增施氮肥能够缓解因细胞分裂素合成减少导致的小麦荧光参数的降低。外源喷施6-BA对两品种小麦内源激素含量影响显著。喷施外源6-BA显著提高旗叶玉米素核苷(ZR)含量,生长素(IAA)含量以及N0、N1处理下21—28 d赤霉素(GA3)含量,显著降低脱落酸(ABA)含量,而喷施外源洛伐他汀后,4种内源激素含量变化与以上结果相反。随着施氮量增加,ZR含量、14—28 d IAA含量随之增加,ABA含量总体呈下降趋势,而7—14 d GA3含量在N1处理下最高。同时,品种、氮肥、激素单一效应、激素与氮肥配合对ZR含量影响显著。另外,6-BA与N1配合对小麦叶绿素含量和抗氧化酶活性的提高效应高于单一喷施6-BA或6-BA与N2配合,激素与氮肥配合施用显著影响叶绿素含量和抗氧化酶活性。与不施氮相比,N1与N2处理下,外源洛伐他汀对叶绿素含量和抗氧化酶系活性的降低幅度减小,即增施氮肥能够缓解因细胞分裂素合成减少引起的两品种小麦光合结构的衰老。花后喷施外源6-BA显著影响千粒重和产量(P0.01),对穗数、穗粒数无显著影响,6-BA与氮肥配合显著影响穗数、穗粒数、千粒重以及产量,6-BA与N1配合显著提高产量及其构成因素。【结论】细胞分裂素与氮肥配合能够明显改善小麦的光合性能,6-BA与适量氮肥配合施用对小麦光合性能的提高效应显著高于单一喷施6-BA或6-BA与过量氮肥相配合,光合性能的改善显著提高两品种的产量。     

玉米衰老相关基因在2个杂交种及其亲本中的表达分析

以玉米杂交种先玉335和郑单958及其亲本为材料,分析了叶片和子粒在授粉后不同时期衰老相关基因表达的变化规律。结果表明,2个叶绿体绿色保持基因SGR1和SGR2在杂交种先玉335和郑单958及其亲本穗三叶中的表达随着授粉后时间的延长整体上呈现上升趋势,但是先玉335的基因表达量要高于郑单958。叶片衰老诱导基因SAP1在叶片发育过程中上调表达,SAP2则表现出先增后减的趋势。以不同生长发育时期的子粒为材料,分析了子粒发育中衰老相关基因SAG1,SAG2和SAG3的表达规律。结果表明,SAG1基因的表达水平随着子粒的发育而降低,而SAG2和SAG3基因则在子粒的成熟过程中表达量呈现上升的趋势。     

Effect of two-week heat stress during grain filling on stem reserves,senescence, and grain yield of European winter wheat cultivars

To examine the extent to which heat stress during grain filling impacts on the development and yield of winter wheat (Triticum aestivum L.), a 3-year field experiment was conducted on a loess soil with high water holding capacity in the North German Plain. Thirty-two mostly European winter wheat cultivars were exposed to heat stress in a mobile foil tunnel with maximum air temperatures of 45.7, 45.4, and 47.2°C in 2015, 2016, and 2017, respectively. The 14-day post-anthesis heat stress treatment caused an average 57.3% grain yield reduction compared to a close-by non-stressed control. The proportion of green crop area after the heat stress phase varied from 7% to 98% in 2016 and from 37% to 94% in 2017. The green crop area percentage did not significantly correlate with grain yield, indicating that the delayed senescence of stay-green phenotypes offers no yield advantage under terminal heat stress. The water soluble carbohydrate (WSC) concentration of the stems at crop maturity varied between 6 and 92 g/kg dry matter, showing that the genotypes differed in their efficiency at using the stem carbohydrate reserves for grain filling under heat stress. The stem WSC concentration correlated positively with the beginning of anthesis (r = 0.704; p < .001) but negatively with the grain yield (r = −0.431; p < .05). For heat tolerance breeding, the stem reserve strategy, i. e. the rapid and full exhaustion of the temporary carbohydrate storage therefore seems more promising than the stay-green strategy.     

EFFECT OF NITROGEN FERTILIZATION ON STAY-GREEN AND SENESCENT SORGHUM HYBRIDS IN SAND CULTURE

Nitrogen (N) is vital in the production of N containing compounds in cereal crops. Stay-greenness in sorghum [Sorghum bicolor (L.) Moench] is characterized by the plant's ability to retain water and chlorophyll content at maturity. A greenhouse experiment was conducted to investigate the effect of the genotypic variability between stay-green (BT × 643) and senescent (RT × 7000) hybrids for leaf senescence, dry matter partitioning and N partitioning under three N levels (0.45, 0.90, 1.80 g plant^?1). The stay-green hybrid had a higher percentage green leaf area at 120 DAE with a higher residual N in leaves. The chlorophyll meter (SPAD-502) values for the stay-green hybrid during anthesis (75 and 85 DAE) were higher compared to its senescent counterpart. The results indicated that the expression of stay-green phenotype in sorghum was contingent on a minimum N supply of 1.35 g plant^?1 and associated with greater N acquisition. Nitrogen use and uptake in the stay-green hybrid were higher.     

玉米自交系开花后叶片保绿度衰减特性遗传效应分析

采用ADM遗传模型对不同玉米自交系开花后叶片保绿度衰减特性遗传效应进行研究。结果表明,不同自交系叶片保绿度衰减起始时间(Ts)、最大衰减速率出现时间(TVmax)、最大衰减速率(Vmax)均存在显著的遗传与环境互作效应,自交系叶片保绿度衰减特性在不同环境条件下的表现具有明显差异。利用包括基因型与环境互作效应在内的遗传分析方法,可以更清楚地发现不同亲本叶片保绿度衰减特性的遗传效应差异,有利于筛选出具有较大遗传效应的自交系,用于玉米叶片保绿度性状的改良。     

QTL mapping of maize (Zea mays) stay-green traits and their relationship to yield

A maize genetic linkage map derived from 115 simple sequence repeat (SSR) markers was constructed from an F₂ population. The F₂ was generated from a cross between a stay-green inbred line (Q319) and a normal inbred line (Mo17). The map resolved 10 linkage groups and spanned 1431.0 cM in length with an average genetic distance of 12.44 cM between two neighbouring loci. A total of 14 quantitative trait loci (QTL) were detected for stay-green traits at different postflowering time intervals and identified by composite interval mapping. The respective QTL contribution to phenotypic variance ranged from 5.40% to 11.49%, with trait synergistic action from Q319. Moreover, maize stay-green traits were closely correlated to grain yield. Additional QTL analyses indicated that multiple intervals of stay-green QTL overlapped with yield QTL.     

持绿型小麦冠温特性及其对低氮和高温的适应性

【目的】研究持绿型小麦的冠温特性及其对高温和低氮的适应性,为小麦低温种质资源鉴选提供依据。【方法】以正常栽培条件为对照,对低氮和高温处理下冬小麦旗叶净光合速率、叶绿素荧光参数、茎鞘干物质量、产量、绿叶面积和冠层温度等进行测定。【结果】在不同生长条件下,持绿型小麦豫麦66和潍麦8均表现出较低的冠层温度和较高的绿叶面积。持绿型小麦旗叶的净光合速率及光化学淬灭系数都高于非持绿型小麦,非光化学猝灭系数低于非持绿型小麦,叶片吸收的光能较多地用于光化学反应,而以热能的形式辐射出来的较少。低氮和高温逆境下持绿型小麦茎鞘干物质转运率增加,收获指数提高,经济产量高。【结论】持绿型小麦冠层温度低,叶片衰老延迟,干物质累积多,具有良好的增产潜力,在低氮和高温逆境下更有优势,可用于筛选低温种质资源和创建具有更好生物学性状、高产的优良品种。     

花后干旱胁迫对不同持绿型玉米叶片衰老的影响

[目的]探讨不同持绿型玉米抗旱的生理机制差异,为促进作物持绿性状的应用,指导作物抗旱改良工作提供理论依据。[方法]利用盆栽试验,以正常灌水为对照,对持绿型玉米自交系齐319和早衰型玉米自交系B73在开花后进行1周的干旱胁迫处理,测定与抗旱相关的生理生化指标及叶片衰老特性。[结果]在干旱胁迫下,与早衰型玉米B73相比,持绿型玉米齐319叶片POD和SOD活性均较高。齐319叶片过氧化物酶(POD)和超氧化物歧化酶(SOD)活性较对照分别增加了32.53%和18.84%,B73较对照分别增加了12.79%和10.82%。干旱胁迫使两玉米自交系的叶片丙二醛(MDA)含量较对照都有显著增加,齐319较对照增加了14.23%,B73较对照增加了37.43%。与对照相比,齐319叶片脯氨酸含量增加了57.95%,B73叶片脯氨酸含量增加了43.67%。且在干旱胁迫下,持绿型玉米齐319具有相对较高的净光合速率(P_n)、气孔导度(G_s)和蒸腾速率(T_r),绿叶面积、叶片保绿度、光合色素含量均高于早衰型玉米B73,而蛋白水解酶活性以及叶绿素水解酶活性均低于早衰型玉米B73。[结论]持绿型玉米齐319在干旱胁迫条件下表现出较强的抗旱性。