高粱苗期耐盐性转录组分析和基因挖掘

【目的】探究高粱耐盐胁迫响应机制,挖掘高粱耐盐胁迫基因,为高粱耐盐育种提供理论基础。【方法】 以高粱感盐品种L甜和耐盐品种石红137为供试材料,采用水培试验。待高粱植株长至三叶一心期,使用2%NaCl溶液对幼苗进行盐胁迫,分别设置0（对照）、1和24 h处理,每个处理3次重复。测定不同处理样品株高、根长、干物重、Na ⁺含量和叶绿素相对含量（SPAD值）,并依托Illumina HiSeq 2000平台进行转录组测序分析。利用FPKM方法计算基因表达量,在差异表达基因检测过程中,将差异表达倍数（fold change）≥2且FDR<0.001作为筛选标准。通过Gene Ontology和KEGG Pathway数据库对参与高粱不同时间盐胁迫差异表达基因进行分析注释。 【结果】 盐胁迫处理对高粱株高、根长、干物重等性状无显著影响,对钠离子含量和SPAD值影响显著。石红137株高、根长、钠离子含量和SPAD值均高于L甜。转录组测序结果鉴定得到已知基因26 628个,新基因866个。石红137中的差异基因数目高于L甜。石红137中,0 h VS 1 h、0 h VS 24 h、1 h VS 24 h三组的差异基因数目分别为375、4 206和3 750个。感盐品种L甜中,0 h VS 1 h、0 h VS 24 h、1 h VS 24 h三组的差异基因数目分别为167、2 534和1 612个。GO分析共获得25个功能注释,分别为光合作用、细胞物质代谢、翻译过程以及激素合成等与盐胁迫相关的差异表达基因。KEGG分析发现盐胁迫1 h表达差异基因富集在植物激素信号转导途径,涉及脱落酸（abscisic acid,ABA）、生长素（auxin,AUX）、细胞分裂素（cytokinin,CTK）、赤霉素（gibberellins,GS）、乙烯（ethylene,ETH）过程等共71个基因。盐胁迫24 h表达差异基因富集于光合作用相关途径,涉及Lhca、Lhcb、磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase,PPC）、磷酸核酮糖激酶（phosphorylribonucleic kinase,PRK）等20个基因。类黄酮生物合成代谢途径差异可能是引起石红137和L甜的耐盐能力差异的原因之一,花青素还原酶（anthocyanidin reductase,ANR）和黄酮醇合成酶（flavonol synthase,FLS）参与类黄酮生物合成途径。【结论】 高粱的盐胁迫过程是一个复杂的生物过程,依赖于多个基因在复杂网络中的平衡表达。盐胁迫条件下,高粱应对环境刺激受到激素信号转导和光合作用的控制。类黄酮生物合成途径在耐盐品种中起到了重要作用。     

延安市高粱新品种引进试验

为了筛选出适合延安市种植的高粱品种,从山西省和辽宁省引入优良高粱品种开展了品种比较试验,并按照最适播种条件进行统一播种和管理。结果表明,综合抗性、产量和品质等因素,晋杂25、辽粘6号、辽粘3号、辽2297和龙杂产量较高,抗性较强,适合进一步试验推广。     

Increasing protein content and digestibility in sorghum grain with a synthetic biology approach

Despite great genetic diversity, sorghum grain consistently suffers from poor protein digestibility. The physicochemical packaging of protein bodies which consist of protease-resistant β- and γ-kafirin is considered a major obstacle. A synthetic β-kafirin gene, which shares the endosperm-specific promoter and signal peptide with the native β-kafirin gene (Sobic.009G001600.1), was transformed into sorghum inbred line Tx430. The gene was modified with ten additional proteolytic sites. These sites were designed to be amenable to cleavage by pepsin and/or chymotrypsin proteinases. Five independent transgenic lines were regenerated by microprojectile transformation. Notably, considerably more protein was observed in the peripheral endosperm of transgenic lines under scanning electron microscopy. Microscopy revealed invaginated or irregularly shaped protein bodies in the endosperm of transgenic lines. Grains of transgenic lines contained 11–37% more protein, which was 11–21% more pepsin digestible and 7–25% more chymotrypsin digestible than Tx430. Additionally, the abundant synthetic β-kafirin protein (5.6% of total protein) was detected by mass spectrometry data analysis in the transgenic line 9-1. Field-grown homozygous transgenics retained higher protein content, larger seed size and no reduction in grain number per plant. The results illustrated that plant synthetic biology could play an important role in improving sorghum nutritional value.     

Individual polyphenolic profiles and antioxidant activity in sorghum grains are influenced by very low and high solar UV radiation and genotype

Sorghum is becoming more widely recognised around the world as a valuable crop for its polyphenol antioxidant health-promoting properties and adaptation to harsh environments. The antioxidant capacity of diverse polyphenols in sorghum grain can vary with climatic conditions and genotype. To explore this further, the potential role of UV radiation on the profile and concentration of polyphenols was investigated in six diverse sorghum genotypes in a controlled environment facility using natural UV and visible radiation under either UV-transmitting or UV-blocking treatments. The polyphenol content and antioxidant activity were significantly reduced under the UV-blocking treatment, with contents of individual polyphenols differing among the genotypes. This information is valuable for sorghum breeders enabling them to select genotypes and UV growth conditions to produce sorghum grain with target levels of polyphenols and antioxidant capacity for human foods to meet the nutritional and health needs of different consumers.     

Diversity,plant growth-promoting traits,and biocontrol potential of fungal endophytes of Sorghum bicolor

The diversity of fungal endophytes in Sorghum bicolor was investigated in samples collected from 10 different geographical regions of Karnataka state, India. A total of 360 endophytes were isolated from leaf, stem, and root tissues and were assigned to 26 fungal species based on morphology and molecular characterization using ITS sequences. All the endophytes belonged to the phylum Ascomycota. The diversity (Shannon H, 2.57; Simpson_1-D, 0.92) and species richness (Margalef's, 4.68; Menhinick, 3.61) were found to be higher for the endophytes isolated from leaf tissues. The species evenness of the endophytic assemblage was strongly influenced by tissue type, followed by geographical location. The biocontrol potential of isolated endophytes was evaluated against economically destructive sorghum grain mould pathogens such as Fusarium thapsinum, Epicoccum sorghinum, Alternaria alternata, and Curvularia lunata using the dual culture method. Biocontrol potential was exhibited by 26 endophytic isolates, of which Trichoderma asperellum recorded broad-spectrum activity against target pathogens, followed by E. nigrum and A. longipes. Most (82%) endophytes showed plant growth-promoting traits. Biosynthesis of indole-3-acetic acid (IAA) was observed in 84% of isolates, and phosphate solubilization, siderophore production, and cellulase activity was observed in 69%, 23%, and 27% of isolates, respectively. Seeds treated with T. asperellum exhibited a significantly higher seed vigour index (2096), germination percentage (94%), and yield under greenhouse and field conditions. The results were substantiated by the confocal microscopy analysis, which clearly demonstrated the colonization of treated endophyte in root tissues. The present study reveals an ecofriendly approach to explore T. asperellum in sorghum disease management.     

贵州省不同年代糯高粱品种（系）农艺性状演变分析

以贵州省不同年代酿酒用糯高粱品种（系）为研究对象,采用随机区组试验设计,对其农艺性状、产量构成以及抗性差异进行分析,结果表明：随着年代更替,贵州省酿酒用糯高粱单产逐步提高。新品种（红缨子、黔高8号）较老品系（黑壳糯、红壳糯）平均单产增幅25%以上;穗粒数、穗粒重增加显著,增幅分别为51.7%、62.7%;主穗一、二级枝梗数增加幅度分别为19.8%、62.7%;株高降低24.9%,茎粗增加9.5%;节间数增加0.9个,节间长缩短13.3cm;单株叶面积和叶面积指数增加显著;倒伏率、发病率下降显著,成穗率明显提升。各指标相关性分析显示,产量与穗粒数、穗粒重,穗粒数与主穗一、二级枝梗数呈极显著正相关;产量与株高、倒伏率极显著负相关,与茎粗正相关,相关系数分别为-0.981、-0.970和0.928,株高、茎粗与倒伏率相关显著,相关系数分别为0.964和-0.910;产量与叶面积指数正相关,与发病率显著负相关。总的结果表明,主穗一、二级枝梗数的增加提高了糯高粱的穗粒数,植株株高的降低、茎粗的增加促进了抗倒性的提升,抗病性的提升保证了后期叶片光合作用的持续进行,最终提高了糯高粱产量。     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

种植密度与行距对秋闲田饲用甜高粱草产量的影响

为了秋闲田饲用甜高粱丰产丰收,探索其高产栽培技术。采用列区试验设计与软件IBM.SPSS.Statistics.v22 的LSD法,对秋闲田饲用甜高粱的种植密度与播种行距进行了单株生产力及其草产量的比较试验。结果表明：种植密度与行距对秋闲田饲用甜高粱单株生产力、草产量均具有重要影响。明确了秋闲田饲用甜高粱高产配套栽培技术的优化组合：单株生产力最大的优化组合为A1B4,即种植密度为7.50 万株/hm2 与播种行距为40 cm 的配置;获得最高产量的优化组合为A2B4,即种植密度为22.50 万株/hm2与播种行距为40 cm的配置。研究结果为秋闲田种植饲用甜高粱的生产实践提了供理论依据和技术支撑。     

高粱幼叶细胞悬浮系的建立

以高粱无菌苗幼叶为试验材料,对颗粒状胚性愈伤组织的获得、细胞悬浮系的建立及影响悬浮细胞生长的主要因素进行了研究。结果表明：幼叶在MS（Murashige and Skoog）+2mg/L 2,4-D培养基上诱导出的愈伤组织,经2~3次继代筛选后获得了浅黄色、颗粒状胚性愈伤组织;胚性愈伤组织接种于液体培养基中,于25±1℃、黑暗条件下,经45~60d的悬浮震荡培养建立了高质量的细胞悬浮系,细胞生长曲线呈“S”型,细胞密度可达6.45×10 ⁵~5.08×10 ⁶个/mL以上,活细胞率可达72.76%,近圆细胞率可达87.50%;培养基的基本成分、激素种类和水平对悬浮细胞生长状态有很大的影响,相比1/2MS培养基,MS培养基为适宜的培养基,2,4-D对保持细胞系的稳定增殖至关重要,适宜的浓度为1mg/L;培养液中加入0.5mg/L KT或6-BA会造成悬浮液褐化,影响悬浮细胞的生长;最适宜的细胞悬浮培养基为L2培养基（MS+1mg/L 2,4-D,30g/L蔗糖,pH5.8）,震荡培养转速为110~120r/min,继代周期为7d,新旧培养基的接种比例为2∶1。     

农杆菌介导高粱遗传转化的相关因素优化

为建立一个稳定的高粱遗传转化体系,以高粱RHMC品种为试验材料,采用内含有P ^CAMBIA3301质粒载体的根癌农杆菌EHA105介导的方法研究了高粱遗传转化的相关因素。结果表明,外植体预培养2d,GUS表达效果最好;在侵染液和共培养基中加入200μmol/L乙酰丁香酮时,GUS表达效果最好;侵染液浓度OD₆₀₀值的适宜范围为0.8~1.0;农杆菌侵染愈伤组织的适宜时间为5~10min;共培养的适宜时间为2d。初步建立了一套高粱遗传转化体系,为今后高粱转化提供一些参考。