小麦-簇毛麦6VS/6AL易位染色体对小麦农艺性状的影响

南京农业大学细胞遗传研究所选育的小麦－簇毛麦6VS/6AL易位系在6VS上携有Pm21基因,用它作抗源已选育出一批高抗白粉病的新品系和新品种。为了研究6VS/6AL易位染色体对普通小麦农艺性状的影响,本研究选用由不同生态类型的推广品种与6VS/6AL易位系经过杂交回交选育的11个高代品系(种)及其轮回亲本和3份涉及6VS/6AL的高代分离品系以及5个F2群体,对产量、株高、穗长、穗粒数、穗粒重和千粒重等农艺性状进行比较分析。结果表明,6VS/6AL易位染色体对后代的小穗数、穗粒数、穗粒重和产量等农艺性状没有表现出明显的影响,对穗长和千粒重表现出一定的正向效应。多数6VS/6AL衍生品系的株高与亲本相比有所增加,但在同一组合的不同品系之间表现出一定的差异,在育种过程中通过选择能够改变增高趋势。6VS/6AL易位系对白粉病免疫,并且遗传稳定,对小麦的抗病育种是很有潜力的抗源亲本。     

小麦—簇毛麦染色体代换系、易位系特异蛋白组分的双向电泳比较分析

利用双向电泳技术,对栽培小麦(AABBDD)、染色体代换系(6V/6A)、易位系(6VS/6AL)、(6VS/6DL)和簇毛麦(VV)的叶片全蛋白进行了比较研究。在栽培小麦、代换系和两个易位系中检测到超过350个蛋白组分,它们的分子量范围是10~110 KD,等电点在4.5～8.6之间。栽培小麦、6V/6A、6VS/6AL、与6VS/6DL之间的双向电泳谱型极为相似,但与簇毛麦不同。在代换系、两个易位系和簇毛麦中检测到了特异蛋白组分16 KD/pI5.0,而在栽培小麦中未检测到该组分,这些结果表明16 KD/pI5.0蛋白可能定位于簇毛麦V染色体短臂上。     

从小麦-簇毛麦易位系TAC文库中筛选Hv-S/TPK基因

本实验室已经通过基因芯片技术筛选到一个白粉菌诱导后上调表达的抗病相关基因Hv-S/TPK, 并获得了它的全长cDNA序列。利用Hv-S/TPK的特异引物筛选小麦-簇毛麦6VS/6AL易位系基因组可转化人工染色体(Transformation-competent artificial chromsome, TAC)文库, 获得了阳性TAC单克隆, 并进一步获得了含有Hv-S/TPK cDNA序列的5160 bp(GenBank Accession No. EU153366)的亚克隆。对亚克隆的序列分析结果表明, Hv-S/TPK基因在起始密码子和终止密码子之间有3个内含子和4个外显子, 4个外显子序列与簇毛麦上已得到的Hv-S/TPK的cDNA序列100%同源。对起始密码子上游序列分析结果表明, 该基因的调控序列中, 含有W-Box、OCS-element等与抗病相关的元件。以TAC克隆为探针与小麦-簇毛麦6VS/6AL易位系有丝分裂中期染色体进行荧光原位杂交(Fluorescence in situ hybridization, FISH), 结果表明含有Hv-S/TPK基因的TAC克隆来自于簇毛麦。     

小麦—冰草T7PL·7AL罗伯逊易位系的分子细胞学鉴定

冰草是小麦遗传改良的重要野生近缘植物之一,有目标的导入冰草外源优异基因是拓宽小麦遗传基础的有效途径。前期研究表明:小麦-冰草衍生系II-23(2n=38W+6P)由19对小麦染色体(缺少4B和7A)和3对冰草染色体(2P、4P和7P)组成。本研究报道从II-23的回交后代中分离鉴定出1个自发易位系7-20。基因组原位杂交(GISH)鉴定表明7-20是一个整臂易位系;经非变性荧光原位杂交(ND-FISH)检测发现,小麦的7A染色体发生易位;进一步利用小麦7A染色体特异SSR标记以及冰草7P染色体特异STS标记对7-20易位系中的外源易位片段大小以及易位染色体的组成进行鉴定,确定7-20为T7PL·7AL罗伯逊易位系(Robertsonian translocation line)。对该易位系与小麦品种Fukuhokomugi构建的BC1F2和BC2F1世代分离群体进行田间农艺性状考察,发现该易位系阳性株系和阴性株系在有效分蘖数和千粒重性状上无显著差异,在株高上表现为阳性材料显著低于阴性材料,但同时出现穗粒数下降的现象。总之,本研究表明易位系7-20为T7PL·7AL罗伯逊易位,该创新材料不仅为后续利用断裂—融合机制创制出更多的补偿易位材料提供了理论依据,而且也为今后向小麦中转移冰草优异基因提供了重要的中间桥梁材料。     

从小麦-簇毛麦易位系TAC文库中筛选Hv-SITPK基因

本实验室已经通过基因芯片技术筛选到一个白粉菌诱导后上调表达的抗病相关基因Hv-S／TPK,并获得了它的全长cDNA序列。利用Hv-S／TPK的特异引物筛选小麦．簇毛麦6VS／6AL易位系基因组可转化人工染色体（Transformation-competent artificial chromsome,TAC）文库,获得了阳性TAC单克隆,并进一步获得了含有Hv-S／TPK cDNA序列的5160bp（GenBank Accession No．EU153366）的亚克隆。对亚克隆的序列分析结果表明,Hv-S/TPK基因在起始密码子和终止密码子之间有3个内含子和4个外显子,4个外显子序列与簇毛麦上已得到的Hv-S／TPK的cDNA序列100％同源。对起始密码子上游序列分析结果表明,该基因的调控序列中,含有W-Box、OCS-element等与抗病相关的元件。以TAC克隆为探针与小麦．簇毛麦6VS／6AL易位系有丝分裂中期染色体进行荧光原位杂交（Fluorescence in situ hybridization,FISH）,结果表明含有Hv-S／TPK基因的TAC克隆来自于簇毛麦。     

小麦3个被白粉菌诱导基因表达的分析

含有抗白粉病基因Pm2 1的小麦 簇毛麦 6VS/6AL易位系在接种白粉菌后 ,叶片无任何病症。应用mRNA差异显示技术从小麦 簇毛麦 6VS/ 6AL易位系分离到 3个叶绿体蛋白基因片段 ,它们是TaD5、TaD2 3和TaD33,3个基因片段分别与小麦叶绿体基因rbcL ,拟斯卑尔脱山羊草叶绿体RNA聚合酶α亚基基因rpoA和大麦 1,5 二磷酸核酮糖羧化酶活化酶基因(Rubiscoactivase ,RcaA2 )同源性达 97%、98%和 88%。据此推测TaD5、TaD2 3和TaD33分别是 6VS/ 6AL易位系中的rbcL、rpoA和 1,5 二磷酸核酮糖羧化酶活化酶基因的片断。Northern分析表明这 3个叶绿体基因的表达在白粉菌诱导下得到增强。叶绿体基因组含有胸腺嘧啶重复区是在mRNA差异显示中克隆到叶绿体基因组基因的原因     

用克隆池PCR法筛选小麦-簇毛麦易位系6VS/6AL基因组TAC文库获得抗病基因候选克隆

用根据抗病基因保守区设计的一对简并性引物,从小麦－簇毛麦易位系6VS／6AL cDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点（Nucleotide binding site,NBS）结构特点的DNA片段克隆N7。从小麦－簇毛麦易位系6VS／6AL基因组TAC（Transformation-competent artificial chromosome,TAC）文库的22块96孔板提取所有2112个克隆池（每个池含约1000个克隆）的质粒,再根据N7的核苷酸序列设计一对特异引物,用克隆池PCR（pooled PCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。研究结果表明克隆池PCR法对克隆数目巨大的基因组文库的筛选很有效。     

小麦中国春-Synthetic 6x代换系的光合速率与产量性状研究

以中国春(Chinese Spring-Synthetic 6x)代换系及其亲本为材料,通过测定旗叶光合速率在各生育时期的变化,研究外源染色体对光合作用的效应;通过观测产量性状,分析外源染色体对小麦产量性状的调控效应.结果表明,从孕穗到灌浆期,5A、5B代换系叶片的光合速率显著高于中国春,说明来自Synthetic 6x的5A、5B染色体上可能携有改良光合特征的相关有利基因;5A、5B代换系的每穗粒数、单穗粒重、千粒重均显著或极显著高于中国春,说明来自Synthetic 6x的5A、5B染色体上可能携有改良每穗粒数、单穗粒重、千粒重有关的有利基因,研究结果可以为小麦的遗传育种提供理论依据.     

Preparation of 6-deoxy-6-fluorocellulose

6-Deoxy-6-fluorocellulose was prepared from cellulose 2,3-diacetate (1) or cellulose 2,3-dibenzoate (2) in various solvents, and was characterized by ¹⁹F and ¹³C NMR measurements. The best product, having ds of 0.95 at C-6 and 0.04 at C-3, was prepared from cellulose 2,3-dibenzoate in nitrobenzene. Other combinations of starting material and solvent gave a lower (≈ 0.8) ds of fluorine at C-6 and higher (≈ 0.12) at C-2 or C-3. Substitution at C-2 was observed when the combination of 1 and 1,4-dioxane, or 2 and chloroform was used. The products substituted at C-2 by fluorine were relatively resistant to acid hydrolysis.     

Facile synthesis of 6-amino-6-deoxycellulose

6-Amino-6-deoxycellulose (4) was synthesized from cellulose by three reaction steps, namely bromination at C-6, displacement of bromine by azide ion, and reduction of the azide group to amino group, in 67% overall yield. The 13C NMR spectrum of compound 4 supports the expected structure for 6-amino-6-deoxycellulose. The degree of substitution of compound 4 was 0.96.     

New 6-butylamino-6-deoxycellulose and 6-deoxy-6-pyridiniumcellulose derivatives with highest regioselectivity and completeness of reaction

This paper investigates the nucleophilic substitution (S(N)) reactions of tosylcellulose with butylamine and pyridine, respectively. The S(N) reactions of tosylcellulose 1 (DS(Total) 2.02; DS(C-6) 1.0) with butylamine carried out at 25, 50, 75 and 100 degrees C in both dimethyl sulfoxide (DMSO) and pure butylamine showed that the regioselectivity of substitution at C-6 of cellulose is temperature dependent: the highest regioselectivity at C-6 can be reached at 25 and 50 degrees C; substitution at C-2 also occurred at 75 and 100 degrees C. The substitution speed in pure butylamine is greater than that in the presence of DMSO. A complete and regioselective substitution at C-6 with a DS of 1.0 was obtained under the conditions of 50 degrees C, 40 h in butylamine. The substitution reactions of 1 with pyridine carried out at 25, 50, 75 and 100 degrees C for 24h in DMSO did not occur. In contrast to this the S(N) reactions done in pure pyridine showed that a temperature- and steric-dependent, regioselective substitution took place at C-6 at temperatures from 25 to 145 degrees C. The highest regioselectivity and completeness at C-6 can be obtained at 100 degrees C for 90 h, whereas at 145 degrees C substitution also occurs at C-2. The results were proved by 1H NMR and 13C NMR spectroscopy.     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Cytotoxic activity of 6-alkynyl- and 6-alkenylpurines

6-Alkynyl- and 6-alkenylpurines have been screened for cytotoxic activity against a human chronic myelogenous leukemia cell line; K-562 cells using a [(3)H]-thymidine incorporation assay. Most alkynes displayed cytotoxicity comparable to, or better than, the known anticancer drugs 6-mercaptopurine and fludarabine. The 6-alkenylpurines, which are promising plant growth stimulators and 15-lipoxygenase inhibitors, exhibited only low toxicity.     

Synthesis and Properties of 6-Deoxy-6-mercapto-d-glucosamine

6-Deoxy-6-mcrcapto-α-d-glucosamine hydrochloride was synthesized from N-anisylidene-1,3,4-tri-O-acetyl-6-O-tosyl-β-d-glucosamine, and some of its properties were compared with those of α-d-glucosamine hydrochloride.     

Preparation of 6-azafulleroid-6-deoxy-2,3-di-O-myristoylcellulose

6-Azafulleroid-6-deoxy-2,3-di-O-myristoylcellulose (3) was synthesized from 6-azido-6-deoxycellulose (1) by two reaction steps. The myristoylation of compound 1 with myristoyl chloride/pyridine proceeded smoothly to give 6-azido-6-deoxy-2,3-di-O-myristoylcellulose (2) in 97.0% yield. The reaction of compound 2 with fullerene (C₆₀) was carried out by microwave heating to afford compound 3 in high yield. It was found from FT-IR, ¹³C NMR, UV–vis, differential pulse voltammetry (DPV), SEC analyses that compound 3 was the expected C₆₀-containing polymer. Consequently, maximum degree of substitution of C₆₀ (DS_C60) of compound 3 was 0.33.     

Syntheses of (+/-)-methyl 6'alpha-demethyl-6'alpha-cyanoabscisate and (+/-)-methyl 6'alpha-demethyl-6'alpha-methoxycarbonylabscisate

New abscisic acid analogs possessing a cyano or methoxycarbonyl group at the 6'alpha-position of methyl abscisate were synthesized by regioselective hydrocyanation. These compounds had weak activity in the rice second leaf sheath elongation test.