Cloning and Functional Analysis of Lycopene ε-Cyclase (IbLCYe) Gene from Sweetpotato, Ipomoea batatas (L.) Lam.

This paper reported firstly successful cloning of lycopene ε-cyclase （lbLCYe） gene from sweetpotato, lpomoea batatas （L.） Lam. Using rapid amplification of cDNA ends （RACE）, lbLCYe gene was cloned from sweetpotato cv. Nongdafu 14 with high carotenoid content. The 1 805 bp cDNA sequence oflbLCYe gene contained a 1236 bp open reading frame （ORF） encoding a 411 amino acids polypeptide with a molecular weight of 47 kDa and an isoelectric point （pI） of 6.95. IbLCYe protein contained one potential lycopene ε-cyclase domain and one potential FAD （flavinadenine dinucleotide）/NAD（P） （nicotinamide adenine dinucleotide phosphate）-binding domain, indicating that this protein shares the typical characteristics of LCYe proteins. The gDNA oflbLCYe gene was 4 029 bp and deduced to contain 5 introns and 6 exons. Real-time quantitative PCR analysis revealed that the expression level of IbLCYe gene was significantly higher in the storage roots of Nongdafu 14 than those in the leaves and stems. Transgenic tobacco （cv. Wisconsin 38） expressing [bLCYe gene accumulated significantly more ^-carotene compared to the untransformed control plants. These results showed that lbLCYe gene has an important function for the accumulation of carotenoids of sweetpotato.

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, lpomoea 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 781xsusceptible 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 Nspl, 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.

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.

Cloning and Characterization of Eukaryotic Translation Initiation Factor 4E (eIF4E) Gene Family in Ipomoea batatas L. (Lam) for Understanding Hexaploid Sweetpotato-Virus Interactions

Characterization of genes related to sweetpotato viral disease resistance is critical for understanding plant-pathogen interactions, especially with feathery mottle virus infection. For example, genes encoding eukaryotic translation initiation factor (eIF)4E, its isoforms, eIF(iso)4E, and the cap-binding protein (CBP) in plants, have been implicated in viral infections aside from their importance in protein synthesis. Full-length cDNA encoding these putative eIF targets from susceptible/resistant and unknown hexaploid sweetpotato (Ipomoea batatas L. Lam) were amplified based on primers designed from the diploid wild-type relative Ipomoea trifida consensus sequences, and designated IbeIF4E, IbeIF(iso)4E and IbCBP. Comparative analyses following direct-sequencing of PCR-amplified cDNAs versus the cloned cDNA sequences identified multiple homeoalleles: one to four IbeIF4E, two to three IbeIF(iso)4E, and two IbCBP within all cultivars tested. Open reading frames were in the length of 696 bp IbeIF4E, 606 bp IbeIF(iso)4E, and 675 bp IbCBP. The encoded single polypeptide lengths were 232, 202, and 225 amino acids for IbeIF4E, IbeIF(iso)4E, and IbCBP, with a calculated protein molecular mass of 26 kDa, 22.8 kDa, and 25.8 kDa, while their theoretical isoelectric points were 5.1, 5.57, and 6.6, respectively. Although the homeoalleles had similar sequence lengths, single nucleotide polymorphisms and multi-allelic variations were detected within the coding sequences. The multi-sequence alignment performed revealed a 66.9% - 96.7% sequence similarity between the predicted amino acid sequences obtained from the homeoalleles and closely related species. Furthermore, phylogenetic analysis revealed ancestral relationships between the eIF4E homeoalleles and other species. The outcome herein on the eIF4E superfamily and its correlation in sequence variations suggest opportunities to decipher the role of eIF4E in hexaploid sweetpotato feathery mottle virus infection.

甘薯(Ipomoea batatas L.)遗传转化几个因素的研究

目的：提高甘薯的遗传转化率,为建立快速高效的甘薯遗传转化体系奠定基础。方法：以甘薯多个优良栽培品种无菌苗茎切段为受体,利用农杆菌LBA4404/pSP10Z做介导,研究了影响甘薯转化的几个因素。结果：接种侵染时间对转化效率影响很大,以2-15min为宜,最高转化率可达32.14%;不同基因型的转化受体之间转化率差别较大;在培养过程中最后30min加入终浓度为50μmol/L AS的接种菌侵染的外植体,转化率较对照提高了2.02倍;共培养培养基中加入终浓度为50μmol/L的AS,转化率较对照组提高了6.11倍;AS终浓度为50μmol/L同时pH为4.8的共培养培养基有利于转化的发生,转化率较对照组提高了1.73倍;共培养培养基中脯氨酸的添加并不能提高转化率。结论：该研究为快速高效甘薯遗传转化体系的建立提供了依据。

甘薯(Ipomoea batatas(L.) Lam.)试管苗种质保存技术研究进展

甘薯种质资源的大田种植保存面临着严重的病毒感染以及病虫害、自然灾害的影响。由茎尖分生组织培养成无病毒苗并进行试管保存,是当前既经济又实用的方法。

甘薯(Ipomoea batatas(L.)lam)不定根根尖细微结构的初步研究

顶端分生组织细胞有高的核质比，胞质中可见前质体和其它细胞器．分化的组织原细胞中，皮层原细胞较中柱原和根冠表皮原细胞内前质体增多明显．组织原细胞内的前质体均匀散布高电子密度基质，并以溢痕方式分裂．淀粉颗粒最先出现在分化早期的皮层细胞和根冠表皮细胞中．较大液泡形成先见于根冠表皮细胞原和中柱内的分化早期细胞．外皮层细胞弦向壁纤维化增厚，径向壁薄，具凯氏带结构．提示甘薯不定根尖早期的外皮层细胞可以分化为既具支持保护作用也适应代谢转运功能的单位．

Analysis of drought tolerance and genetic and epigenetic variations in a somatic hybrid between Ipomoea batatas (L.) Lam. and I. triloba L.

The somatic hybrid KT1 was previously obtained from protoplast fusion between sweetpotato （Ipomoea batatas （L.） Lam.） cv. Kokei No. 14 and its wild relative I. triloba L. However, its genetic and epigenetic variations have not been investigated. This study showed that KT1 exhibited significantly higher drought tolerance compared to the cultivated parent Kokei No. 14. The content of proline and activities of superoxide dismutase （SOD） and photosynthesis were significantly increased, while malonaldehyde （MDA） content was significantly decreased compared to Kokei No. 14 under drought stress. KT1 also showed higher expression level of well-known drought stress-responsive genes compared to Kokei No. 14 under drought stress. Amplified fragment length polymorphism （AFLP） and methylation-sensitive amplified polymorphism （MSAP） analyses indicated that KT1 had AFLP and MSAP band patterns consisting of both parent specific bands and changed bands. Fur- ther analysis demonstrated that in KT1. the proportions of Kokei No. 14 specific genome components and methylation sites were much greater than those of I. triloba. KT1 had the same chloroplast and mitochondrial genomes as Kokei No. 14. These results will aid in developing the useful genes ofI. triloba and understanding the evolution and phylogeny of the cultivated sweetpotato.

Identification and Quantification of Allelochemicals from Selected Sweet Potato (<i>Ipomoea batatas</i>(L.) Lam.) Cultivars

Allelopathic compounds have the potential to inhibit the growth and development of other organisms in a diverse manner ranging from shifting nutrients and enhancing their growth to inflicting diseases. In addition, these compounds influence seedling growth and seed germination of various crops. The goal of this study was to identify and quantify different allelochemicals in various sweet potato cultivars through high-performance liquid chromatography techniques. Selected sweet potato slips (weight: 2.0 - 2.5 grams/slip) were propagated in separate glass tubes filled with 10.0 mL distilled water. Water extract from each glass tube was collected after 2, 4, and 6 weeks after transplanting (WAP) to identify and quantify allelochemical compounds by comparing their peaks with the retention time of standards. Results show that the concentration of allelochemicals in water extract was increased from 2 to 4 WAP but remained constant in the sixth week. Quantitative analysis revealed that the amount of chlorogenic acid was higher in all sweet potato cultivars compared to other allelochemicals. Some sweet potato cultivars, A5 and A39, exhibited higher allelopathy (18.28 - 19.37 ppm/slip) and reduced the height and biomass of Palmer amaranth the most due to the presence of increased concentration of combined allelochemicals, while other cultivars produced lesser allelochemicals (10.90 ppm/slip) and did not reduce the growth of the weed species. Allelopathic sweet potato cultivars high in chlorogenic acid production can effectively suppress Palmer amaranth with minimal dependence on chemicals to manage weeds and harmful pests under sustainable agricultural system.

甘薯(Ipomoea batatas L.Lam.)基因启动子在根癌农杆菌中的作用

作者曾用自行构建的启动子探针型载体ｐＳＵＰＶ４，从甘薯（ＩｐｏｍｏｅａｂａｔａｔａｓＬ．Ｌａｍ．）总ＤＮＡ中克隆到了６５个在大肠杆菌中具有启动基因表达功能的ＤＮＡ片段．现研究发现，甘薯基因启动子片段在根癌农杆菌中也具有启动基因转录的功能，其启动功能的大小与启动子片段在大肠杆菌中启动卡那霉素抗性基因表达活性呈正相关，即原来卡那霉素抗性越高的，ＧＵＳ基因的酶活性越高．利用重组子ｐＩＢ２中插入片段所做的Ｓｏｕｔｈｅｒｎ杂交实验证实，该ＤＮＡ片段来源于甘薯基因组．

AFLP Fingerprinting and Genetic Diversity of Main Sweetpotato Varieties in China

AFLP fingerprinting of the 98 main sweetpotato varieties planted in China has been constructed. Using 17 AFLP primer combinations which were selected from 1 208 primer combinations and generated the most amounts of polymorphic bands, AFLP analysis of the 98 main sweetpotato varieties gave a total of 410 clear polymorphic bands with an average of 24.12 polymorphic bands per primer combination. Each one of the 98 sweetpotato varieties could be clearly distinguished by EcoR I-cta/Mse I-ggc primer combination which generated the most polymorphic bands. AFLP-based genetic distance ranged from 0.0546 to 0.5709 with an average of 0.3799. The dendrogram based on AFLP markers indicated that sweetpotato varieties coming from the same regions or having same parents were clustered in the same groups. Analysis of molecular variance （AMOVA） revealed greater variations within regions （94.08%） than among regions （5.92%）. Thus, the genetic variations mainly existed within regions, while the variations among regions were very low in the tested sweetpotato varieties. Significant genetic variations existed between ＂Northern＂ and ＂Southern＂ sweetpotato varieties when Yangtze River was used as the dividing line.

Transcriptome profiling reveals insights into the molecular mechanism of drought tolerance in sweetpotato

Sweetpotato,Ipomoea batatas(L.) Lam.,is a globally important food crop and usually grown on arid-and semi-arid lands.Therefore,investigating the molecular mechanism of drought tolerance will provide important information for the improvement of drought tolerance in this crop.In this study,transcriptome analysis of the drought-tolerant sweetpotato line Xushu 55-2 was conducted on Illumina HiSeq 2500 platform.A total of 86.69 Gb clean data were generated and assembled into 2 671 693 contigs,222 073 transcripts,and 73 636 unigenes.In total,11 359 differentially expressed genes(DEGs) were identified after PEG6000 treatment,in which 7 666 were up-regulated and 3 693 were down-regulated.Of the 11 359 DEGs,10 192 DEGs were annotated in at least one database,and the remaining 1 167 DEGs were unknown.Abscisic acid(ABA),ethylene(ETH),and jasmonic acid(JA) signalling pathways play a major role in drought tolerance of sweetpotato.Drought-inducible transcription factors were identified,some of which have been reported to be associated with drought tolerance and others are unknown in plants.In addition,7 643 SSRs were detected.This study not only reveals insights into the molecular mechanism of drought tolerance in sweetpotato but also provides the candidate genes involved in drought tolerance of this crop.

Cloning and Characterization of a Salt Tolerance-Associated Gene Encoding Trehalose-6-Phosphate Synthase in Sweetpotato

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes： trehalose-6-phosphate synthase（TPS） and trehalose-6-phosphate phosphatase（TPP）. In the present study, a TPS gene, named IbTPS, was first isolated from sweetpotato（Ipomoea batatas（L.） Lam.） cv. Lushu 3 by rapid amplification of cDNA ends（RACE）. The open reading frame（ORF） contained 2 580 nucleotides encoding 859 amino acids with a molecular weight of 97.433 kDa and an isoelectric point（pI） of 5.7. The deduced amino acid sequence showed high identities with TPS of other plants. Real-time quantitative PCR analysis revealed that the expression level of IbTPS gene was significantly higher in stems of Lushu 3 than in its leaves and roots. Subcellular localization analysis in onion epidermal cells indicated that IbTPS gene was located in the nucleus. Transgenic tobacco（cv. Wisconsin 38） plants over-expressing IbTPS gene exhibited significantly higher salt tolerance compared with the control plant. Trehalose and proline content was found to be significantly more accumulated in transgenic tobacco plants than in the wild-type and several stress tolerance related genes were up-regulated. These results suggest that IbTPS gene may enhance salt tolerance of plants by increasing the amount of treahalose and proline and regulating the expression of stress tolerance related genes.

Molecular Cloning and Functional Characterization of a Salt Tolerance-Associated Gene IbNFU1 from Sweetpotato

Iron-sulfur cluster biosynthesis involving the nitrogen fixation（Nif） proteins has been proposed as a general mechanism acting in various organisms.NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses.Based on the EST sequence selected from salt-stressed suppression subtractive hybridization（SSH） cDNA library constructed with a salt-tolerant mutant LM79,a NFU gene,termed IbNFU1,was cloned from sweetpotato（Ipomoea batatas（L.） Lam.） via rapid amplification of cDNA ends（RACE）.The cDNA sequence of 1 117 bp contained an 846 bp open reading frame encoding a 281 amino acids polypeptide with a molecular weight of 30.5 kDa and an isoelectric point（pI） of 5.12.IbNFU1 gene contained a conserved Cys-X-X-Cys motif in C-terminal of the iron-sulfur cluster domain.The deduced amino acid sequence had 66.08 to 71.99% sequence identity to NFU genes reported in Arabidopsis thaliana,Eucalyptus grandis and Vitis vinifera.Real-time quantitative PCR analysis revealed that the expression level of IbNFU1 gene was significantly higher in the roots of the mutant LM79 compared to the wild-type Lizixiang.Transgenic tobacco（cv.Wisconsin 38） plants expressing IbNFU1 gene exhibited significantly higher salt tolerance compared to the untransformed control plants.It is proposed that IbNFU1 gene has an important function for salt tolerance of plants.

Enhanced Stem Nematode Resistance of Transgenic Sweetpotato Plants Expressing Oryzacystatin-I Gene

Enhanced stem nematode resistance of transgenic sweetpotato （cv. Lizixiang） was achieved using Oryzacystatin-I （OCI） gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.

A geranylgeranyl pyrophosphate synthase gene, Ib GGPS, increases carotenoid contents in transgenic sweetpotato

Geranylgeranyl pyrophosphate synthase(GGPS) plays an important role in the biosynthesis of carotenoids. In a previous study, the IbGGPS gene was isolated from a sweetpotato, Ipomoea batatas(L.) Lam., line Nongdafu 14 with high carotenoid contents, but its role and underlying mechanisms in carotenoid biosynthesis in sweetpotato were not investigated. In the present study, the IbGGPS gene was introduced into a sweetpotato cv. Lizixiang and the contents of β-carotene, β-cryptoxanthin, zeaxanthin and lutein were significantly increased in the storage roots of the IbGGPSoverexpressing sweetpotato plants. Further analysis showed that IbGGPS gene overexpression systematically upregulated the genes involved in the glycolytic, 2-C-methyl-D-erythritol-4-phosphate(MEP) and carotenoid pathways,which increased the carotenoid contents in the transgenic plants. These results indicate that the IbGGPS gene has the potential for use in improving the carotenoid contents in sweetpotato and other plants.

Identification of Differentially Expressed Genes in Sweetpotato Storage Roots Between Kokei No.14 and Its Mutant Nongdafu 14 Using PCR-Based cDNA Subtraction

The contents of earotenoids in the storage root of sweetpotato, Ipomoea batatas （L.） Lam. vary dramatically among different cultivars. However, so far little is known about the regulation of carotenoids synthesis in sweetpotato. In our laboratory, we identified a novel sweetpotato mutant, Nongdafu 14, which is a homogenous mutant derived from the wild type Kokei No. 14. The contents of carotenoids in the storage root of Nongdafu 14 were analyzed using high performance liquid chromatography （HPLC）, and it was found that the amount of carotenoids, [3-carotene, lutein and zeaxantion, three major types of earotenoids in sweetpotato storage roots, increased 2-26 folds in Nongdafu 14 compared to Kokei No. 14. Suppression subtractive hybridization （SSH） was used to identify genes that were differentially expressed in Nongdafu 14, and a differentially expressed eDNA library was constructed using the eDNA of Nongdafu 14 storage roots as tester and that of Kokei No. 14 storage roots as driver. Out of the 1 530 clones sequenced, we identified 292 nonredundant ESTs. GO and KEGG analyses of these differentially expressed ESTs indicated that diverse metabolism pathways were affected and candidate genes involved in regulation of carotenoids synthesis are suggested.

甘薯淀粉含量的QTL定位

本研究以高淀粉甘薯品种漯徐薯8号(母本)和低淀粉甘薯品种郑薯20(父本)杂交得到的F1分离群体,选择其中240个单株为材料,以该群体所构建的分子连锁图谱为基础,以2008年和2009年所测定的淀粉含量为指标,采用复合区间作图法并利用QTLMapper2.0软件分析了甘薯淀粉含量的QTL。实验结果表明,检测到了1个主效位点,位于父本郑薯20的Z31连锁群上a02b40.02ds**和a08b37.03fs*两个标记之间,QTL的加性效应为-0.73,解释表型变异的7.70%。定位甘薯淀粉含量相关的QTL,将为发掘与淀粉含量相关的基因奠定基础。

HPLC法测定药用甘薯西蒙Ⅰ号中咖啡酸的含量

目的 建立测定西蒙 号叶中有效成分含量的方法 ,比较不同年份的西蒙 号叶中咖啡酸的含量差异。方法 采用 HPL C对西蒙 号叶中咖啡酸的含量进行测定。结果 咖啡酸在 10 .33～ 16 5 .2 8μg/ m L 具有良好的线性关系 ,平均回收率为 99.38% ,RSD=1.36 % (n=6 )。结论 该方法灵敏、准确、专属性强 ,适用于西蒙 号药材中咖啡酸的测定。

甘薯叶绿体rbcL基因的克隆与序列分析

根据烟草、水稻和菠菜叶绿体的全基因组序列设计引物,以甘薯的叶绿体基因组DNA为模板,PCR扩增包含甘薯叶绿体rbcL完整基因(GenBank登录号为AY942199)在内的一段序列。序列分析表明:此片段的全长为1627bp,包括1443bp的编码区序列在内,推测编码480个氨基酸,同时构建了此片段的限制性酶切图谱。相似性比较显示,此基因编码区序列与烟草、菠菜、小麦、水稻、玉米、矮牵牛、紫花苜蓿、拟南芥、莨菪、葡萄以及甜菜的rbcL基因核苷酸的同源性为85%～98%,氨基酸的同源性为92%～95%。