基于甘薯毛状根的外源多基因表达系统的建立

用携带植物高效表达载体pCAMBIA1304+的"解除武装"的重组C58C1工程菌转化甘薯新品种"渝苏303"无菌苗的真叶,50个外植体中的36个长出了毛状根,诱导率达72%.PCR捡测证实发根型质粒pRiA4和表达型质粒pCAMBIA1304+均能整合到甘薯毛状根基因组内,共转化率达36%.建立了基于发根农杆菌介导的甘薯毛状根的高效外源基因表达系统,为利用甘薯毛状根快速验证基因功能和将多个外源基因导入甘薯,实现其代谢工程及开展分子育种奠定了基础.     

粘毛黄芩毛状根外源基因表达系统的建立

用携带植物高效表达载体pCAMBIA1304+和发根型质粒pRiA4,解除武装的重组C58C1工程菌感染粘毛黄芩(Scutellaria viscidula Bunge.)无菌苗的幼茎、子叶、叶片,诱导毛状根的表达,以建立基于粘毛黄芩毛状根的高效外源基因表达系统。幼茎毛状根诱导率最高,达88.9%。PCR扩增检测粘毛黄芩毛状根单克隆基因组中的rolC和hygr基因片段,结果表明,pRiA4和pCAMBIA1304+均能整合到粘毛黄芩毛状根的基因组内,共转化率达28.1%。     

转基因何首乌毛状根的研究进展

何首乌毛状根体系因为遗传稳定、生长迅速的特点,在次生代谢产物合成和提取方面具有优势。本文概述了何首乌毛状根体系的诱导和培养过程、影响何首乌毛状根生长及其次生代谢产物积累的因素,并就何首乌毛状根在生物转化以及次生代谢工程方面的应用前景进行了展望。     

转基因何首乌毛状根的研究概况及其应用前景

何首乌毛状根体系因为遗传稳定、生长迅速的特点,在次生代谢产物合成和提取方面具有优势。本文概述了何首乌毛状根体系的诱导和培养过程、影响何首乌毛状根生长及其次生代谢产物积累的因素,并就何首乌毛状根在生物转化以及次生代谢工程方面的应用前景进行了展望。     

茶树毛状根的诱导

利用几种不同发根农杆菌株系感染茶树外植体，得到茶树毛状根。通过筛选，找出对其诱导效率最高的一个株系是15834，并证实了乙酰丁香酮对诱导具有促进作用。     

冬凌草毛状根体系的建立

用发根农杆菌(Agrobacterium rhizogenes)ATCC15834、ACCC10060、C58C1菌株对冬凌草无菌幼苗的叶片、茎段进行侵染,而后通过共培养的方法诱导其产生冬凌草毛状根,从而建立有效的冬凌草毛状根培养体系。通过PCR进一步检测T-DNA是否成功导入冬凌草毛状根中。通过用发根农杆菌ATCC15834、ACCC10060、C58C1菌株对冬凌草外植体进行侵染,能够成功诱导产生冬凌草毛状根,从而对冬凌草毛状根培养体系进行建立,在一定程度上为冬凌草毛状根的大规模培养奠定基础。     

黄秋葵再生体系的建立及毛状根的诱导

[目的]建立黄秋葵的再生体系并诱导其毛状根.[方法]以黄秋葵无菌苗的叶片和茎段为外植体,建立黄秋葵的体细胞再生体系,并利用发根农杆菌A4侵染黄秋葵叶片,诱导毛状根.[结果]愈伤组织的最佳诱导培养基为MS＋ 1.0 mg/L 6-BA ＋0.5 mg/L NAA时;最佳体细胞胚发生培养基为MS ＋0.5 mg/L NAA;毛状根最佳诱导条件为叶片预培养48h,侵染时间6min.[结论]该研究为成功构建黄秋葵遗传转化体系和研究黄秋葵中的次生代谢产物奠定了基础.     

黄秋葵再生体系的建立及毛状根的诱导

[目的]建立黄秋葵的再生体系并诱导其毛状根。[方法]以黄秋葵无菌苗的叶片和茎段为外植体,建立黄秋葵的体细胞再生体系,并利用发根农杆菌A4侵染黄秋葵叶片,诱导毛状根。[结果]愈伤组织的最佳诱导培养基为MS+1.0 mg/L 6-BA+0.5 mg/L NAA时;最佳体细胞胚发生培养基为MS+0.5 mg/L NAA;毛状根最佳诱导条件为叶片预培养48 h,侵染时间6 min。[结论]该研究为成功构建黄秋葵遗传转化体系和研究黄秋葵中的次生代谢产物奠定了基础。     

甘薯贮藏根淀粉代谢相关基因表达分析

甘薯淀粉含量高,是一种重要的粮食作物和工业原料。然而,甘薯贮藏根发育过程中淀粉代谢途径相关基因昼夜周期变化规律尚不完全清楚。为探讨甘薯贮藏根发育过程中淀粉代谢相关基因昼夜周期变化规律,本研究以4个不同淀粉含量的甘薯品种为试验材料,采用qRT-PCR法对淀粉代谢相关基因全天的表达模式进行分析。结果表明:IbAGPa、IbSS、IbGBSSⅠ、IbSBEⅠ、IbSBEⅡ、Ibα-amylase基因的表达均呈现先升高后降低的趋势。淀粉合成相关基因(IbAGPa、IbSS、IbGBSSⅠ、IbSBEⅠ、IbSBEⅡ)的表达高峰出现在12时到15时,淀粉降解相关基因(Ibα-amylase)的表达高峰出现在15时之后。在不同淀粉含量的甘薯品种中,淀粉代谢相关基因的表达在整个昼夜周期内都发生周期性的变化,且表达趋势相似。本研究进一步丰富了我们对甘薯贮藏根淀粉代谢调控机制的认识。     

山豆根毛状根培养体系的建立与优化

为探索定向培养山豆根植物细胞及器官获取有效药用成分的新途径,为山豆根有效药用成分的大规模生产奠定基础,采用共培养法研究发根农杆菌R1601感染诱导山豆根无菌苗子叶产生毛状根,并在此基础上从不同培养液、不同蔗糖浓度和外源激素对毛状根生长的影响方面筛选山豆根毛状根生长的最佳培养液。结果表明:利用发根农杆菌R1601感染山豆根无菌苗子叶16min,在MS+AS 100μmol/L的固体培养基上培养可诱导产生毛状根,且诱导率最高,达66.67%;1/2 MS+IAA 0.5mg/L+5%蔗糖的培养液最有利于山豆根毛状根的生长。     

SSR fingerprinting of 203 sweetpotato (Ipomoea batatas (L.) Lam.) varieties

Simple sequence repeat (SSR) markers have been shown to be a powerful tool for varieties identification in plants. However, SSR fingerprinting of sweetpotato varieties has been a little reported. In this study, a total of 1294 SSR primer pairs, including 1215 genomic-SSR and 79 expressed sequence tag (EST)-SSR primer pairs, were screened with sweetpotato varieties Zhengshu 20 and Luoxushu 8 and their 2 F₁ individuals randomly sampled, and 273 and 38 of them generated polymorphic bands, respectively. Four genomic-SSR and 3 EST-SSR primer pairs, which showed good polymorphism, were selected to amplify 203 sweetpotato varieties and gave a total of 172 bands, 85 (49.42%) of which were polymorphic. All of the 203 sweetpotato varieties showed unique fingerprint patterns, indicating the utility of SSR markers in variety identification of this crop. Polymorphism information content (PIC) ranged from 0.5824 to 0.9322 with an average of 0.8176. SSR-based genetic distances varied from 0.0118 to 0.6353 with an average of 0.3100 among these varieties. Thus, these sweetpotato varieties exhibited high levels of genetic similarity and had distinct fingerprint profiles. The SSR fingerprints of the 203 sweetpotato varieties have been successfully constructed. The highly polymorphic SSR primer pairs developed in this study have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction in sweetpotato and other plants.     

Accumulation and Gene Expression of Anthocyanin in Storage Roots of Purple- Freshed Sweet Potato [Ipomoea batatas （L.） Lam] Under Weak Light Conditions

Anthocyanidin in plants, an important pigment, is of great interest to researchers, consumers, and commercial entities due to its physiological functions. Anthocyanin content and mRNA levels of anthocynin biosynthesis genes were investigated in storage root of different purple-fleshed sweet potatoes （PFSP） genotypes to understand the regulation mechanism of anthocyanin under weak light conditions. Anthocyanin content, its amount of accumulation, and the expression of CHS, DFR, F3H, GT, and ANS genes in the PFSP storage root under weak light conditions were studied. The results demonstrated that the anthocyanin content of the treatments was decreased and was obviously lower than that of the control until 30 days after shading in Ayamurasaki, while it was lower than that of the control from the beginning of shading in Jishu 18. Their accumulation rates of both treatmeants were lower than its control before 10-20 d of shading in Jishu 18, while those of Ayamurasaki weren＇t in their treatments. This indicated that Jishul 8 is more sensitive to light as compared to Ayamuraska. Under the different weak light conditions, mRNA levels for ibCHS, ibF3H, ibDFR, and ibANS were obviously decreased, while the expression of ibGT was increased. These results indicated that anthocyanin content was regulated by light at the mRNA levels and the enzymatic level in sweet potato. Therefore, the development dynamic response to anthocyanin content varied in different genotypes of PFSP, and mRNA levels of anthocyanin biosynthesis were inhibited under the weak light condition.     

脱毒对甘薯形态特征及产量的影响

[目的]探讨脱毒甘薯生长发育特性和增产效果,选育出适合当地农业生产的脱毒甘薯。[方法]以"一窝红"和"卢选1号"2个甘薯品种为研究材料,对其整个生育过程进行调查,对各关键时期植株生长发育的形态指标进行了测定和研究。[结果]脱毒在很大程度上影响了甘薯植株的生长发育,一些形态及产量指标如蔓长,茎、叶鲜重,块根直径,块根重量,茎分支数,结薯数和单株产量都发生显著变化。生育前期脱毒甘薯植株的生长优势表现在地上部,后期优势表现在以产量形成为主的地下根部。[结论]脱毒甘薯的增产效果明显,因此应用脱毒苗是当前解决病毒病的最好方法。     

Identification of QTLs for Starch Content in Sweetpotato(Ipomoea batatas(L.) Lam.)

Sweetpotato（Ipomoea batatas（L.）Lam.）breeding is challenging due to its genetic complexity.In the present study,interval mapping（IM）and multiple quantitative trait locus（QTL）model（MQM）analysis were used to identify QTLs for starch content with a mapping population consisting of 202 F1 individuals of a cross between Xushu 18,a cultivar susceptible to stem nematodes,with high yield and moderate starch,and Xu 781,which is resistant to stem nematodes,has low yield and high starch content.Six QTLs for starch content were mapped on six linkage groups of the Xu 781 map,explaining 9.1-38.8%of the variation.Especially,one of them,DMFN₄,accounted for 38.8%of starch content variation,which is the QTL that explains the highest phenotypic variation detected to date in sweetpotato.All of the six QTLs had a positive effect on the variation of the starch content,which indicated the inheritance derived from the parent Xu 781.Two QTLs for starch content were detected on two linkage groups of the Xushu 18 map,explaining 14.3 and 16.1%of the variation,respectively.They had a negative effect on the variation,indicating the inheritance derived from Xu 781.Seven of eight QTLs were co-localized with a single marker.This is the first report on the development of QTLs co-localized with a single marker in sweetpotato.These QTLs and their co-localized markers may be used in marker-assisted breeding for the starch content of sweetpotato.     

Identification of QTLs for Starch Content in Sweetpotato （Ipomoea batatas （L.） Lam.）

Sweetpotato （Ipomoea batatas （L.） Lam.） breeding is challenging due to its genetic complexity. In the present study, interval mapping （IM） and multiple quantitative trait locus （QTL） model （MQM） analysis were used to identify QTLs for starch content with a mapping population consisting of 202 F1 individuals of a cross between Xushu 18, a cultivar susceptible to stem nematodes, with high yield and moderate starch, and Xu 781, which is resistant to stem nematodes, has low yield and high starch content. Six QTLs for starch content were mapped on six linkage groups of the Xu 781 map, explaining 9.1-38.8% of the variation. Especially, one of them, DMFN 4, accounted for 38.8% of starch content variation, which is the QTL that explains the highest phenotypic variation detected to date in sweetpotato. All of the six QTLs had a positive effect on the variation of the starch content, which indicated the inheritance derived from the parent Xu 781. Two QTLs for starch content were detected on two linkage groups of the Xushu 18 map, explaining 14.3 and 16.1% of the variation, respectively. They had a negative effect on the variation, indicating the inheritance derived from Xu 781. Seven of eight QTLs were co-localized with a single marker. This is the first report on the development of QTLs co-localized with a single marker in sweetpotato. These QTLs and their co-localized markers may be used in marker-assisted breeding for the starch content of sweetpotato.     

甘薯Ipomoea batatas(L.) Lam.茎蔓多糖提取技术研究

通过正交实验研究了甘薯茎蔓多糖水提过程中提取温度、时间和提取用水量对提取物得率的影响,选出了较为理想的提取条件,即加40倍水,在100℃下浸提25min,得率4 82%。通过棕色环法、蒽酮—硫酸法、Fe hling试剂法、双缩脲法证实提取物为多糖。     

超声波辅助提取甘薯根颈多糖

甘薯根颈是甘薯生产中的大宗副产物,是提取功能性多糖的廉价资源。通过正交试验,对甘薯根颈多糖超声波辅助提取的影响因素进行了筛选和优化。结果表明：料液比、超声波频率、浸提温度、超声波功率、超声浸提时间等因素对提取率影响较大,较为合理的提取方案为：料液比1：30,超声波频率26kHz,超声波功率350W,浸提温度60℃,浸提时间30min。以此优化方案提取3次,提取物的平均提取得率为4.95%。通过棕色环试验、蒽酮-硫酸法、Fehling试剂法、双缩脲法证实提取物为多糖。     

Development of SRAP Markers Linked to a Gene for Stem Nematode Resistance in Sweetpotato, Ipomoea batatas (L.) Lam.

Sequence-related amplification polymorphism (SRAP) markers closely linked to stem nematode resistance gene were developed in sweetpotato, Ipomoea batatas (L.) Lam. Using bulked segregant analysis (BSA), 200 SRAP primer combinations were screened with the resistant and susceptible bulked DNA from the 196 progenies of an F1 single-cross population of resistant parent Xu 781×susceptible parent Xushu 18, 77 of them showed polymorphic bands between resistant and susceptible DNA. Primer combinations detecting polymorphism between the two bulks were used to screen both parents and 10 individuals from each of the bulks. The results showed that primer combination A9B4 produced 3 specific bands in the resistant plants but not in the susceptible plants, suggesting that the markers, named Nsp1, Nsp2 and Nsp3, respectively, linked to a gene for stem nematode resistance. Primer combination A3B6 also produced a SRAP marker named Nsp4 linking to the resistance gene. Amplified analysis of the 196 F1 individuals indicated that the genetic distance between these markers and the resistance gene was 4.7, 4.7, 6.3, and 9.6 cM, respectively.