拟南芥硫苷生物合成相关基因的组织和胁迫诱导表达谱的全基因组分析

【目的】分析拟南芥中硫代葡萄糖苷（Glucosinolatcs,简称硫苷）生物合成相关基因在不同组织器官、不同发育时期及不同生物和非生物胁迫处理条件下的表达谱,为筛选控制硫苷合成的关键基因、解析硫苷的生物合成规律及其在植物抗逆胁迫过程中的作用奠定基础。【方法】利用AtGenExpress和PLEXdb中的10组表达谱芯片数据和2组转录组数据分析拟南芥中硫苷生物合成途径82个结构基因和调控基因的时空表达特性及其受生物和非生物胁迫的表达模式;采用Real-time PCR技术对10个硫苷合成途径的关键结构基因和调控基因的表达谱进行验证;并利用STRING v10软件分析硫苷生物合成途径相关基因的表达模式的相关性。【结果】组织表达特性分析结果表明,拟南芥硫苷生物合成相关基因在营养器官和生殖器官中的表达模式差异较大,相关基因在营养器官中具有较高的表达量,在生殖器官中的表达量则较低,表明硫苷主要在营养组织中合成;其中脂肪族硫苷合成相关基因在茎、叶等地上营养组织中的表达量较高,而吲哚族硫苷合成相关基因则在根、茎、叶等地上和地下组织中均有较高的表达,且基因在同一组织的不同发育时期的表达量通常具有差异,表明基因的表达还具有时空特异性;荧光定量PCR分析结果证明10个硫苷合成途径代表基因的表达谱与芯片数据的结果保持一致;胁迫表达分析结果表明,脂肪族和吲哚族硫苷合成相关基因的表达均受到部分生物和非生物胁迫因子的诱导表达,其中吲哚族硫苷合成相关基因更易受到丁香假单胞杆菌（Pseudomonas syringae）、甘蓝链格孢菌（Alternaria brassicicola）、桃蚜（Myzus persicae）等生物和低温（4℃）、高盐、高温（38℃）等非生物胁迫因子的诱导表达;共表达分析结果表明,脂肪族和吲哚族硫苷合成分支途径内相关调控基因与结构基因的表达模式的相关性较高。【结论】拟南芥硫苷生物合成相关基因主要在营养组织中高表达,在种子中低表达;植物中存在一个硫苷转运系统,负责将营养组织中合成的硫苷转运到种子中贮藏;硫苷合成相关基因的表达受到病原菌、虫害、高温等逆境因子的影响,其中吲哚族硫苷合成相关基因更易受到生物和非生物胁迫因子的诱导表达,说明吲哚族硫苷可能对植物抗性更为重要。     

油菜籽饼粕中硫苷的酶解条件优化及降解产物分析

【目的】研究影响油菜籽饼粕中硫代葡萄糖苷在黑芥子酶作用下定向转化为异硫氰酸酯的因素,确定最佳酶解条件,探索外加黑芥子酶对硫代葡萄糖苷降解产物组分和含量的影响,为油菜籽饼粕的综合利用提供基础。【方法】测定4种反应体系中黑芥子酶活性,确定酶解反应缓冲体系。以油菜籽饼粕中异硫氰酸酯总量为指标,单因素结合正交设计研究不同料液比、时间、温度、pH、抗坏血酸添加量对外加黑芥子酶降解油菜籽饼粕中硫代葡萄糖苷转化为异硫氰酸酯的影响,优化工艺参数,获得最佳酶解条件。采用二氯甲烷为提取剂,真空浓缩得到油状降解产物,利用气相色谱-质谱（GC-MS）联用鉴定外加黑芥子酶和内源黑芥子酶酶解产物成分,分析产物相对含量。【结果】4种缓冲体系中,在柠檬酸-磷酸盐缓冲液中测得黑芥子酶活性最高,在相同条件下,外加黑芥子酶的活性显著高于内源黑芥子酶活性;外源黑芥子酶添加量大于油菜籽饼粕量的20%,异硫氰酸酯生成量无显著增加,选择外源黑芥子酶添加量为油菜籽饼粕量的20%;正交优化得到最佳酶解条件为：料液比1﹕15,pH为6.0,反应温度为50℃,抗坏血酸添加量为0.005 mg•g-1,反应时间为1 h,异硫氰酸酯含量达到1.799 mg•g-1,是油菜籽饼粕内源黑芥子酶降解硫代葡萄糖苷生成异硫氰酸酯产物总量的7.6倍。外加黑芥子酶生成异硫氰酸酯含量显著高于内源黑芥子酶降解硫苷生成异硫氰酸酯含量,但两者异硫氰酸酯含量随反应条件变化趋势一致。GC-MS鉴定外加黑芥子酶有7种酶解产物,相对含量在产物的67%以上,含有1种腈类,2种噁唑烷酮和4种异硫氰酸酯。产物中异硫氰酸酯为烯丙基异硫氰酸酯、丁烯基异硫氰酸酯、环戊基异硫氰酸酯和苯乙基异硫氰酸酯;其中丁烯基异硫氰酸酯的含量最高,占酶解产物总量的26.77%。腈类产物为苯丙腈;2种噁唑烷酮为致甲状腺肿素和2,1-苯丙噁唑烷酮。内源黑芥子酶降解硫代葡萄糖苷的产物中检测到4种产物且含量相对较少。【结论】外加黑芥子酶能有效增加油菜籽饼粕中异硫氰酸酯生成量,确定了油菜籽饼粕中硫代葡萄糖苷的最佳降解条件。鉴定出降解产物的主要成分,异硫氰酸酯有4种,其中丁烯基异硫氰酸酯含量最高。     

Measurement of the Glucosinolate Content in Rapeseed Using the TRUBLUGLU Meter

A simple and inexpensive method for the estimation of total glucosinolate (GSL) Content In rapeseed is described. It employs a newly developed portable reflectance instrument, the TRUBLUGLU Meter. The assay is based on the selective hydrolysis of Gsls By Endogenous myrosinase at pH 9, followed by measurement of the released glucose using Clinistix strips. The meter, which is calibrated prior to use with a standard glucose solution, can display a digital readout in units of either mM glucose or μol Gsl/g Seed.     

Alkenyl Glucosinolate Levels in Androgenic Populations of Brassica napus

The levels of individual and total alkenyl glucosinolates in seeds of microspore-derived spontaneous diploid plants from low by high and low by low glucosinolate parent crosses were examined to assess the utility of haploidy in canola breeding. The distributions of lines in the populations supported previous proposals that alkenyl glucosinolate levels are under multigenic control. Levels of all of the individual glucosinolates were positively correlated and were significantly reduced in canola-quality material in comparison to rapeseed-quality material. The populations of microspore-derived lines from low × high crosses were skewed to high glucosinolate levels but the population from a low × low glucosinolate cross had a greater proportion of low glucosinolate lines. The former observations can be explained in terms of the dominance of genes for high glucosinolate levels in Brassica napus. The present findings contradict previous reports that androgenic lines have higher glucosinolate content than the parents and in fact, haploidy may select for low glucosinolate lines when crosses between low glucosinolate parents are used.     

Genetic analysis of total glucosinolate in crosses involving a high glucosinolate Indian variety and a low glucosinolate line of Brassica juncea

Analysis of the glucosinolate content and composition by high‐pressure liquid chromatography indicated that varieties of Brassica juncea bred and grown in India have a high glucosinolate content characterized by the presence of 2‐propenyl (allyl) and 3‐butenyl as the major and 4‐pentenyl as the minor fractions. In contrast, the B. juncea germplasm from other countries is characterized by the presence of 2‐propenyl as the major glucosinolate fraction, trace amounts of 3‐butenyl and a total lack of the 4‐pentenyl types. In order to transfer the low glucosinolate trait to Indian B. juncea, the inheritance of total glucosinolates was investigated using doubled haploid (DH) populations derived from F1 (DH₁) and BC₁ (BC₁DH) of a cross between ‘Varuna’ (the most widely cultivated high glucosinolate variety of India) and ‘Heera’ (a non‐allyl type low glucosinolate line). A total of 752 DH₁ and 1263 BC₁DH gave rise to seven and 11 low glucosinolate (containing less than 18 μmol/g seed) individuals, respectively. On the basis of the frequency of the low glucosinolate individuals, the total glucosinolate was found to be under the control of seven genes. There was presence of both allyl and non‐allyl types in DH₁ and BC₁DH low‐glucosinolate individuals and absence of 3‐butenyl glucosinolate in some of the BC1DH low glucosinolate individuals, indicating segregation for these fractions in the population. The size of the segregating DH population proved to be crucial for precise determination of the number of genes controlling the trait. Because of the large number of genes involved, incorporation of low glucosinolate trait in Indian B. juncea should be approached through doubled haploid (DH) breeding.     

单、双低甘蓝型油菜胞质雄性不育系及恢复系选育研究

研究以秦油2号出现的不育株（简称陕ZA）为基本材料，与自育和引进的国内外单双低（低芥酸、低硫么）油菜品种（系）为父本．采用刚交、回交的方法育成了一批单双低甘蓝型油菜胞质雄性不育系及其相应的保持系，并实现了“三系”配套。在不育系的选育中发现，无论是单低或是双低不育系，其不育性的差异主要表现在不育度上，一些表现彻底不育．一些表现有微粉、这主要决定于保持系；且在这些不同的不育系之间，不同的目交方式之间所得到的自交亲和指数不同。同时还进行了新的单双低恢复系的选育研究，已取得了初步进展。     

西兰花中硫代葡萄糖苷的提取及抑菌试验初报

采用甲醇法提取西兰花中硫代葡萄糖苷,利用重量法测定其中硫代葡萄糖苷的含量,得出硫代葡萄糖苷最佳提取条件,并对硫代葡萄糖苷提取液进行酸水解,通过抑菌圈实验,分析了西兰花中硫代葡萄糖苷水解产物异硫氰酸酯类的抑菌性。其结果表明,采用80％甲醇溶液、料液比为10：40（w／v）、80℃为提取的最佳条件。西兰花花蕾中硫代葡萄糖苷的含量最高,达到39．90μmol／g;花茎中为18．28μmol／g：叶中为11．86μmol／g。实验结果表明异硫氰酸酯类对金黄色葡萄球菌（Staphylococcus aureus）、白葡萄球菌（White staphylococcus）、枯草芽孢杆菌（Bacillus subtilis）大肠杆菌（Escherichia coli）的抑菌作用均较明显。     

芥蓝菜薹不同节间芥子油苷组分及含量分析

采用高效液相色谱及液相色谱-质谱联用法,以芥蓝(Brassica alboglabra L.)3个品种‘中花’、‘澄海粗条,和'全年油青’为材料,对菜薹不同节间(菜薹从膨大处到顶端依次平分为4节)芥子油苷组分和含量进行了鉴定和分析.结果表明,除4-甲硫基丁基芥子油苷在全年油青菜薹的第1节中没有检测到、4-羟基-3-吲哚...     

Growth,feeding and thyroxine‐related responses of hybrid striped (sunshine) bass (Morone chrysops ♀ X M. saxatilis ♂) fed de‐oiled carinata (Brassica carinata) meal

De‐oiled (<10 g/kg oil) carinata (Brassica carinata) meal is high (>400 g/kg) in protein, but its use in fish diets will likely be limited mainly by glucosinolates (GLS), sinapine and crude fibre. Two feeding experiments were conducted to determine the response of hybrid striped bass (Morone chrysops ♀ X M. saxatilis ♂, HSB) to inclusion of cold‐pressed carinata meal (CPCM) in diets. Cook extrusion reduced an average of 44% and 57% of GLS and sinapine concentrations in diets, respectively. In experiment 1, diets containing 0.71–2.71 μmoles of GLS and 0.034–0.181 mg of sinapine/g did not significantly (p > .05) reduce feed consumption, utilization or growth, resulting in similar concentrations of thyroxines and consequently no effect on deiodinase enzymes. In experiment 2, there was a significantly reduced (p < .05) feed consumption in fish fed diets containing 5.58–9.52 μmoles of GLS and 0.54–0.75 mg of sinapine/g, resulting in HSB exhibiting lethargic swimming and feeding behaviours, and consequently poor feed utilization and growth. Cook extrusion reduced about half of GLS and sinapine, and HSB tolerated ≤2.71 μmoles of GLS and ≤0.31 mg of sinapine/g of diet without affecting feed consumption and utilization, growth and thyroid metabolism.     

四川生态区甘蓝型油菜自交种和自然种品质测试的比较研究

[研究目的]比较研究四川生态区甘蓝型油菜自交种和自然种粗脂肪、硫苷含量的测试结果有无差异;[方法]对四川生态区861份甘蓝型油菜自交种和自然种用近红外光谱法(NIRS法)进行品质测试;用SPSS统计分析软件对测试结果进行相关和回归分析;[结果]以NIR法测试甘蓝型油菜自然种粗脂肪含量普遍较自交种高,自交种粗脂肪含量与自然种粗脂肪含量差异达1%显著水平,自交种与自然种硫苷含量差异不显著.自交种和自然种粗脂肪、硫苷含量的NIR法测试结果间相关关系、线性回归方程均达1%显著水平,自然种粗脂肪(y1)、硫苷(y2)含量与自交种粗脂肪(x1)、硫苷(x2)含量测试结果间的回归方程分别为y=16.844 0.614x1,y2=-0.620 1.017x2;[结论]在甘蓝型油菜育种中,对粗脂肪的选择应侧重考虑自然种含量,兼顾考虑自交种含量,对硫苷的选择则可同时考虑自然种和自交种含量.