Genome-wide identification and expression analysis of the β-amylase gene family in Ipomoea batatas
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摘要:目的
挖掘甘薯Ipomoea batatas基因组中β−淀粉酶(Beta-amylase)基因家族序列信息，分析结构与功能信息。
方法基于甘薯栽培种‘泰中6号’全基因组测序数据，利用生物信息学分析方法对鉴定到的12个甘薯β−淀粉酶家族成员进行结构域保守性分析、染色体定位、潜在重复基因筛查、保守基序分析和系统进化树构建，利用转录组数据进行低温胁迫下相关基因的表达分析。
结果12个β−淀粉酶基因分布于甘薯第2、4、5、6、11、12、13和14号染色体上，含有8个具有潜在重复关系的基因对。多重比对和功能结构域搜索结果显示，甘薯β−淀粉酶家族的氨基酸序列中含有3个保守性较高的区域和10个保守基序。甘薯与其他物种β−淀粉酶蛋白的系统进化分析结果显示，62个β−淀粉酶家族成员被分为S1~S7等7个亚组，甘薯β−淀粉酶家族成员主要分布在S2、S4、S5、S6以及S7亚组中，且大多与拟南芥、马铃薯以及番茄的β−淀粉酶为同一分支。转录组测序数据分析结果显示，在低温贮藏的过程中有6个甘薯β−淀粉酶基因表达量出现变化，其中‘徐薯15-1’有2个基因上调表达、4个基因下调表达，‘徐薯15-4’仅有2个基因下调表达。
结论β−淀粉酶是一类关键的淀粉水解酶，在甘薯生长发育和薯块贮藏阶段淀粉降解为还原糖的过程中发挥着重要作用，本研究鉴定得到的12个甘薯β−淀粉酶基因序列信息为进一步探讨甘薯β−淀粉酶基因家族的生物学功能提供数据参考。
Abstract:ObjectiveTo mine the sequence information of the β-amylase gene family of Ipomoea batatas genome, and analyze the structure and function of genes.
MethodBased on the whole genome sequence data of I. batatas cultivar ‘Taizhong 6’, the bioinformatic methods were applied to analyze the identified 12 members of the β-amylase gene family and conduct the domain conservation analysis, chromosomal localization, screening of potential duplication genes, conservative motif analysis, phylogenetic tree construction. The gene expression under low temperature stress was analyzed using the transcriptomics data.
ResultTwelve β-amylase genes were located on chromosomes No. 2, 4, 5, 6, 11, 12, 13 and 14 of I. batatas, and eight pairs showed potential duplication relationship. Multiple sequence alignment and functional domain search indicated that there were three highly conserved domains and 10 conservative motifs in the amino acid sequences of I. batatas β-amylase family. Phylogenetic trees of β-amylase proteins in I. batatas and other species showed that 62 β-amylase family members were divided into seven subgroups of S1−S7. The β-amylases of I. batatas were mainly distributed in the subgroups of S2, S4, S5, S6 and S7, most of which belonged to the same branches with Arabidopsis thaliana, Solanum tuberosum and S. lycopersicum. The results of transcriptomics data showed that six β-amylase genes expressed differentially during the low temperature storage period, of which two were up-regulated and four were down-regulated in ‘Xushu 15-1’, while only two genes were down-regulated in ‘Xushu 15-4’.
ConclusionThe β-amylases are a key class of starch hydrolyzing enzymes that play important roles in the degradation of starch into reducing sugars during the process of I. batatas growth, development and tuber storage stages. The sequences of the identified 12 sweet potato β-amylase genes provide data reference for further study on the biological functions of I. batatas β-amylase gene family.
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Keywords:
- Ipomoea batatas /
- β-amylase /
- gene family /
- systematic evolution /
- differential expression analysis
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动物的采食行为是维持机体能量稳态的基础，畜禽生产中获得充足的食物是其生长发育的前提。动物采食量受中枢调控，其中胃肠道状态是决定畜禽食欲的关键部位。揭示饥饿状态下鸡食欲调控的潜在肠−脑轴机制可为如何提高鸡采食量提供理论依据。下丘脑弓状核作为食欲调控中枢[1-2]存在大量的促采食的刺鼠相关蛋白(Agouti-related protein，AgRP)/神经肽 Y(Neuropeptide Y，NPY)神经元和抑采食的前阿片黑色皮质素(Proopiomelanocortin，POMC)/可卡因−苯丙胺调节转录肽(Cocaine and amphetamine regulated transcript，CART)神经元[3-6]。影响动物食欲的因素有很多，遗传、环境因素、机体健康以及肠道充盈状态等均能影响动物采食量[7]。其中胃肠道作为营养物质暂时储存和消化吸收的关键部位，存在大量食欲调控信号[8]。这些食欲调控信号一方面通过血液循环被中枢所感应[2, 9]，另一方面被肠道迷走感觉传入神经元直接感应，经脑干孤束核最终将信号投递至食欲调控中枢，肠道和中枢间的这种信息传递被称为“肠−脑轴” [8, 10]。肠道迷走感觉神经作为假单极双向神经元，位于结状神经节处的胞体分别向中枢孤束核和肠道发出轴突，其中肠道迷走神经末梢存在多种受体感应肠道各种理化信号，例如游离脂肪酸受体2 (FFAR2)、生长激素促分泌素受体(GHSR)、胆囊收缩素受体(CCKR)，以及炎症受体TLR4等 [10-12]。
肠道健康对机体维持高食欲具有重要作用，维持肠道平衡可以维持机体正常食欲，反之肠道菌群紊乱等原因导致肠道健康受损则会引起采食量降低[13-14]，而肠道屏障是肠道发挥其正常生物学功能的重要前提[15-16]，肠道黏膜屏障包括肠上皮细胞及胞间连接，其中，紧密连接是肠上皮细胞间的细胞旁通路的主要屏障，闭合(Occludin)和紧密连接(Claudin)家族成员是影响其功能的主要封闭蛋白，二者与胞浆蛋白相互作用维持紧密蛋白的完整性[17-18]。当肠道出现炎症时，会导致Claudin蛋白结构变化，进而引起肠道屏障功能性障碍，并且受致病菌侵害也会导致肠道屏障通透性增加[19]；动物炎症性肠病会导致肠道隐窝改变、小肠绒毛萎缩或变平以及一系列的形态学变化[20]。大量研究发现间歇性饥饿有助于维持肠道及肠道屏障的完整性[21-22]。
此外，胃肠道中上皮基质和微生物群落共调控生成活性氧，导致生成H2O2；而H2O2是维持正常细胞稳态和生理功能所必需的第二信使[23]。Miller等[24]研究发现，结肠内壁中的细胞会释放H2O2(而非氧气)来限制微生物的生长，H2O2可以协同其他物质在肠道黏膜上形成保护，防止菌群紊乱或肠道炎症对机体造成损伤，并且可以治疗肠道炎症，恢复机体正常生理功能。然而，目前并不清楚短期饥饿是否影响肠道炎症水平和屏障功能、是否被迷走感觉神经所感应。本研究旨在揭示禁食后肠道炎症水平和肠道屏障变化，以及提高食欲的潜在机制，并提供理论基础和试验依据。
1. 材料与方法
1.1 试验动物与试验设计
选用20只1日龄初生黄羽肉鸡[25-28](购于广东省清远市凤翔麻鸡发展有限公司生产基地)，试验前称体质量并排序，随后按配对随机设计的原则将体质量相近的小鼠分为2组：对照组和禁食组，每组10只黄羽肉鸡，正常饲喂饲料至5日龄并采样。采样前12 h，禁食组禁食，对照组正常采食。禁食12 h后收集小肠肠道内容物检测H2O2水平，采集黄羽肉鸡结状神经节(Nodose ganglia，NG)，检测炎症和食欲相关受体的表达；采集十二指肠、空肠和回肠及其肠道黏膜，检测黄羽肉鸡肠道形态、闭锁小带蛋白−1 (Zonula，ZO-1)、闭合蛋白 (Occludens-1，OCC)、紧密连接蛋白(Claudin-1) 以及炎症因子的表达。
1.2 测定指标与方法
1.2.1 小肠肠道内容物
分离小肠，区分十二指肠、空肠和回肠，取部分肠道轻轻挤压，将内容物收集于 2 mL 离心管中，使用过氧化氢测定试剂盒(A064-1-1，南京建成生物工程研究所)检测H2O2水平。
1.2.2 扫描电子显微镜(SEM)
取一段1 cm长的空肠，剪开后平铺，用生理盐水轻轻清洗内容物，而后修剪为5 mm边长的正方形放于保存液中，于4 ℃条件下保存。而后脱水、干燥，进行电镜扫描。
1.2.3 苏木精−伊红(HE) 染色
小肠分离后剪取约 3 cm 空肠中段放于 40 g/L 的多聚甲醛中固定，按照常规方法制作石蜡切片，HE染色，光学显微镜下拍照，然后用Image软件测取肠道绒毛长度(lv)和隐窝深度(dc)，每个切片取 3~5 个视野，取其平均值计算绒毛长度与隐窝深度比值(lv/dc)。
1.2.4 小肠黏膜及 NG 的 RNA 提取、逆转录和荧光定量PCR (q-PCR)
小肠黏膜及NG总 RNA 使用 RNA 提取试剂盒(R4130-02，广州美基生物科技有限公司)和 TRIzol 试剂提取。1 g 总 RNA 按试剂盒说明书用 4× Reverse Transcription Master Mix(EZB-RT2GQ，美国 EZBioscience 生物技术有限公司)逆转录成 cDNA。引物序列见表1，按照2× SYBR Green qPCR Master Mix(A0012-R2，美国 EZBioscience 生物技术有限公司)说明书配制反应体系：10 μL 的体系中含有 5 μL 2× Color SYBR Green qPCR Master Mix、3.6 μL dd H2O、1 μL cDNA、0.4 μL 引物工作液；使用 Applied Biosystems QuantStudio 3 实时 PCR 系统并按照以下程序反应：95 ℃预热 5 min；95 ℃ 10 s，60 ℃ 30 s，循环 40 次。根据对照组 β-actin mRNA 表达进行归一化处理[15]。
表 1 实时荧光定量PCR所用引物Table 1. Primers used for quantitative real-time PCR基因
Gene上游引物序列(5′→3′)
Forward primer sequence下游引物序列(5′→3′)
Reverse primer sequence序列号
Accession numberβ-actin CTGTGCCCATCTATGAAGGCTA ATTTCTCTCTCGGCTGTGGTG L08165 AgRP CTCTTCCCAGGCCAGACTTG GCAGAAGGCGTTGAAGAACC XM_046925680.1 CCKAR AGCTCTTCTGCCAACCTGAT GTGTAGGACAGCAGGTGGAT NM_001081501.2 Claudin-1 TGGAGGATGACCAGGTGAAG TGTGAAAGGGTCATAGAAGG NM_001013611.2 CART CGAGAGAAGGAGCTGATCGA AGAAAGGAGTTGCACGAGGT XM_046937244.1 FFAR2 GCACTCTCTTTATGGCTGCC GGATTCCCTGGTCTTGGTCA XM_040693461.2 IL-1 CCTCCTCCAGCCAGAAAGTG CGGTAGAAGATGAAGCGGGT XM_015297469.3 IL-4 CCCCAGGTGTAGGCTCTAGT ACTCTGTCATTGCTGCTCCC XM_040683457.2 IL-6 ACCCGAGCTCTTTGGTGATG CGTGCCCTCTGTTTGTACCT XM_025143427.3 IL-10 GCTGCCAAGCCCTGTT CCTCAAACTTCACCCTCA NM_001004414.4 GHSR ATTAGTGCTGGCCCCATCTT CGGACCGATGTTCTTCCTCT XM_046923539.1 MC4R AGGGGTCATCATCACATGCA GATGGCCCCTTTCATGTTGG NM_001031514.2 NPY GTGCTGACTTTCGCCTTGTC ATCTCTGCCTGGTGATGAGG NM_205473.2 Occludin TGGAGGAGTGGGTGAAGAAC ATCCTTCCCCTTCTCCTCCT XM_046904540.1 POMC AGAGGAAGGCGAGGAGGAAA GTAGGCGCTTTTGACGATGG XM_046914234.1 TLR-4 GGCTCAACCTCACGTTGGTA AGTCCGTTCTGAAATCCCGT NM_001030693.2 TNF-α TTCTATGACCGCCCAGTT CAGAGCATCCAACGCAAAA XM_046920820.1 NPY2R GGCCATCATCTCCTATGCCT GGAAGCCAACTGACAGCAAA NM_001398092.1 ZO-1 TCATCCTTACCGCCGCATAT GTTGACTGCTCGTACTCCCT XM_046925214.1 1.3 数据统计与分析
所有数据均以平均值±标准误差(Mean±SE)表示。用GraphPad Prism 8.0 软件进行统计分析。采用 t 检验对2组均值进行差异显著性分析。
2. 结果与分析
2.1 禁食后下丘脑内食欲肽相关受体表达变化
通过 q-PCR 检测下丘脑内食欲肽相关基因表达，结果发现，与对照组相比，雏鸡禁食12 h后促采食食欲肽基因AgRP (P<0.05)和 NPY (P<0.01)的 mRNA 相对表达量均显著上调(图1)，提示雏鸡饥饿模型构建成功。
![图 1 黄羽肉鸡禁食12 h后下丘脑内食欲肽相关受体表达的变化]()
图 1 黄羽肉鸡禁食12 h后下丘脑内食欲肽相关受体表达的变化“*”和“**”分别表示差异达到 0.05和0.01的显著水平(t检验)Figure 1. Expression changes of orexin-related receptors in hypothalamus of yellow-feathered broilers after 12 h of fasting“*” and “**” indicate that the difference reaches 0.05 and 0.01 significance levels respectively (t test)2.2 禁食对黄羽肉鸡空肠肠道形态的影响
空肠肠绒毛电镜扫描及分析结果如图2A、3A、3B 所示，观察发现雏鸡禁食12 h 后，同对照组相比空肠肠绒毛表面更加完整，单位面积内绒毛总数更多、受损更少并且排列更加整齐。空肠 HE 染色及分析结果如图2B、3C、3D 所示，与正常采食的雏鸡相比，禁食后雏鸡的隐窝深度和lv／dc均无明显变化，但是对照组绒毛有明显损伤，而禁食组绒毛排列整齐、长度更长。
![图 2 黄羽肉鸡禁食12 h对空肠肠道绒毛形态的影响]()
图 2 黄羽肉鸡禁食12 h对空肠肠道绒毛形态的影响Figure 2. Effects of fasting for 12 h on jejunum intestinal villus morphology of yellow-feathered broilers![图 3 黄羽肉鸡禁食12 h后空肠肠道绒毛形态变化的电镜扫描结果(A、B)和HE 染色结果(C、D)统计]()
图 3 黄羽肉鸡禁食12 h后空肠肠道绒毛形态变化的电镜扫描结果(A、B)和HE 染色结果(C、D)统计Ⅰ：对照组，Ⅱ：禁食组；“*”和“**”分别表示差异达到 0.05和0.01的显著水平(t检验)Figure 3. Statistics of the scanning electron microscopy results (A, B) and HE staining results (C, D) for the morphological changes of jejunum intestinal villi of yellow-feathered broilers after fasting for 12 hⅠ: Control, Ⅱ: Fasting group; “*” and “**” indicate that the difference reaches 0.05 and 0.01 significance levels respectively (t test)2.3 禁食对黄羽肉鸡肠道屏障的影响
由图4 可知，与对照组相比，禁食12 h后雏鸡小肠黏膜中紧密蛋白标志性基因ZO-1和Occludin mRNA的相对表达量均显著上调(P<0.05)，在十二指肠中，Claudin-1 的mRNA相对表达量也显著上调(P<0.05)。
![图 4 黄羽肉鸡禁食12 h后小肠肠道黏膜紧密蛋白的mRNA相对表达量变化]()
图 4 黄羽肉鸡禁食12 h后小肠肠道黏膜紧密蛋白的mRNA相对表达量变化“*”和“**”分别表示差异达到 0.05和0.01的显著水平(t检验)Figure 4. mRNA relative expression changes of intestinal mucosal compact protein in small intestine of yellow-feathered broilers after 12 h fasting“*” and “**” indicate that the difference reaches 0.05 and 0.01 significance levels respectively (t test)2.4 禁食对黄羽肉鸡肠道炎症水平的影响
由图5可知，黄羽肉鸡禁食12 h后，与对照组相比，十二指肠、空肠和回肠黏膜上炎症因子IL-1、IL-6和TNF-α的 mRNA表达量无明显变化，但是空肠黏膜抗炎因子IL-4和IL-10的 mRNA表达量均有显著升高(P<0.01)。并且空肠和回肠内容物中H2O2浓度均有不同程度的增加(图3 D )。
![图 5 黄羽肉鸡禁食12 h后小肠炎症因子mRNA相对表达量及H2O2浓度变化]()
图 5 黄羽肉鸡禁食12 h后小肠炎症因子mRNA相对表达量及H2O2浓度变化图D中，DU：十二指肠，Anterior JE：空肠前段，Middle JE：空肠中段，Posterior JE：空肠后段，Anterior IL：回肠前段，Posterior IL：回肠后段；“*”和“**”分别表示差异达到 0.05和0.01的显著水平(t检验)Figure 5. Changes in mRNA relative expressions of intestinal inflammatory factors and H2O2 concentrations in yellow-feathered broilers after 12 h of fastingIn figure D, DU: Duodenum, Anterior JE: Anterior jejunum, Middle JE: Middle jejunum, Posterior JE: Posterior jejunum, Anterior IL: Anterior ileum , Posterior IL: Posterior ileum; “*” and “**” indicate that the difference reaches 0.05 and 0.01 significance levels respectively (t test)2.5 禁食后雏鸡NG内受体表达的变化
由图6A 可知，与对照组相比，雏鸡禁食12 h后 NG 内肠道炎症因子IL-4的受体基因IL-4R的mRNA相对表达量显著上调(P<0.01)。由图6B 可知，禁食组雏鸡NG内食欲相关受体基因的mRNA相对表达量有所增加，其中FFAR2和神经肽2受体(NPY2R)表达量增加显著(P<0.01)。
![图 6 黄羽肉鸡禁食12 h后结状神经节内炎症(A)与食欲(B)相关受体mRNA相对表达量]()
图 6 黄羽肉鸡禁食12 h后结状神经节内炎症(A)与食欲(B)相关受体mRNA相对表达量“**”表示差异达到0.01的显著水平(t检验)Figure 6. mRNA relative expression of inflammation-related (A) and orexin-related (B) receptors in nodose ganglia of yellow-feathered broilers after 12 h of fasting“**” indicates that the difference reaches 0.01 significance level (t test)3. 讨论与结论
已有研究发现，特异性激活下丘脑弓状核AgRP神经元显著提高动物采食量[29]，诱导肥胖发生[30]，而消除AgRP神经元则会导致厌食症[31]。因此，本研究首先检测了下丘脑弓状核食欲肽表达变化，结果发现短期禁食后黄羽肉鸡下丘脑 AgRP/NPY表达显著上调(P ＜ 0.05)，而POMC有下降趋势(P = 0.07)，提示黄羽肉鸡饥饿模型构建成功。
肠道健康对机体维持高食欲具有重要作用，而肠道炎症则会影响肠道代谢水平、破坏微生物平衡[32]以及肠道屏障的完整性[33]，甚至会影响中枢神经系统中神经肽的分泌，大量研究发现间歇性饥饿有助于维持肠道及肠道屏障的完整性[21-22]。据报道，胃肠道中上皮基质和微生物群落共调控生成活性氧，导致H2O2形成；而H2O2是维持正常细胞稳态和生理功能所必需的第二信使[23]。本试验通过检测小肠不同肠段内容物的H2O2浓度发现，短期禁食导致禁食组空肠和回肠内容物中H2O2浓度均有不同程度的增加，推测饥饿状态下肠道可能通过生成适量H2O2维持肠道稳定。为进一步验证这一假设，我们通过电镜扫描、HE染色以及q-PCR结果发现，短期禁食并未对肠道形态造成损伤，且由于缺少食物影响，肠道绒毛排列更加紧凑整齐。我们推测，机体短期禁食后尚未引发肠道疾病，并且在肠道饥饿状态下，因肠道营养物质缺乏，机体可能出于自我保护机制防止肠道毒素等有害因子进入机体，从而紧密连接增强，即肠道物理屏障增强，且抗炎因子的表达增加，降低空肠损伤比例，避免肠道受损，以抵抗禁食给机体带来的不良影响，维持肠道正常的生理功能，这对维持较高食欲至关重要。
大量研究报道，肠道食欲调控信号不仅可以通过血液信号被中枢所识别，还可以被肠道迷走感觉传入神经元直接感应，经肠−脑轴最终将信号投递至食欲调控中枢[10-12]。本试验结果发现，与对照组相比，雏鸡禁食12 h后结状神经节内IL-4受体基因的mRNA相对表达量显著上调，和肠道黏膜抗炎因子表达变化相对应；提示机体在饥饿状态下，可能通过提高肠道抗炎能力以及增强物理屏障来抵抗由禁食所导致的轻微炎症，维持肠道健康。
此外，结状神经节内食欲相关受体基因的mRNA相对表达量有所增加，其中FFAR2和NPY2R表达量增加显著(P ＜ 0.01)，推测黄羽肉鸡饥饿后由于AgRP和NPY表达量增加[4]，并且FFAR2和NPY2R表达增加，二者将肠道饥饿信号传递至中枢神经系统，提高动物食欲进而促进采食量增加。
综上所述，饥饿可引起肠道抗炎因子水平升高，并维持肠道屏障完整性，同时促进迷走感觉神经末梢抗炎因子受体表达，最终引起食欲增强。
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图 1 甘薯β−淀粉酶保守结构域分析
蓝色阴影部分表示该位点氨基酸保守性为100%，红色阴影部分表示该位点氨基酸保守性在70%以上；统计时各区域内由空位所代替的序列不纳入计算；Region I画横线部分表示flexible loop，Region II“*”标注处表示催化活性位点，Region III画横线部分表示Inner loop
Figure 1. Analysis of the conserved domains of β-amylase in Ipomoea batatas
The blue shaded part indicates that the amino acid conservation of the site is 100%, and the red shaded part indicates that the amino acid conservation of the site is over 70%; The series replaced by vacancies in each region are excluded in the calculation; The horizontal line in Region I represents the Flexible loop, the “*” in Region II shows the catalytic active site, and the horizontal line in Region III denotes the Inner loop
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图 2 甘薯β−淀粉酶基因家族染色体定位及潜在重复关系分析
Figure 2. Chromosomal localization and potential duplication relationship of the β-amylase gene family in Ipomoea batatas
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图 3 甘薯β−淀粉酶蛋白序列保守基序分析
Figure 3. Conservative motif analysis of protein sequences of Ipomoea batatas β-amylases
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图 4 甘薯与拟南芥、马铃薯、番茄、大豆、水稻、玉米以及大麦的β−淀粉酶系统进化分析
S1~S7为7个亚组
Figure 4. Phylogenetic analysis of β-amylases in eight species, i.e. Ipomoea batatas, Arabidopsis thaliana, Solanum tuberosum, Solanum lycopersicum, Glycine max, Oryza sativa, Zea mays and Hordeum vulgare
S1−S7 are seven subgroups
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图 5 甘薯β−淀粉酶基因低温胁迫下不同贮藏时间的表达分析
Figure 5. Expression analysis of β-amylase gene in Ipomoea batatas at different storage time under low temperature stress
表 1 甘薯β−淀粉酶基因序列信息1)
Table 1 The sequence information of β-amylase gene in Ipomoea batatas
基因名称
Gene name外显子
Exon链
Strand长度/bp
LengthCM008332.1-snap.12826 45177690-45177779; 45176575-45176835; 45176174-45176392; − 798 45175888-45176091; 45175660-45175665; 45174982-45174999 CM008334.1-snap.6488 29784343-29784606; 29784692-29784856; 29785241-29785504; + 909 29786090-2978630 CM008335.1-snap.6154 26986968-26987510; 26986519-26986728; 26986320-26986430; − 1 647 26985282-26986064 CM008336.1-snap.4559 14557970-14558494; 14560135-14560344; 14560435-14560551; + 1 701 14560653-14561441; 14561707-14561766 CM008336.1-snap.4659 14866852-14867396; 14867730-14867844; 14868220-14868330 + 771 CM008341.1-snap.11840 40434808-40434870; 40433953-40434363; 40433695-40433859; − 1 521 40432752-40433015; 40432065-40432280; 40431587-40431988 CM008342.1-snap.286 1511146-1511589; 1512051-1512279; 1512390-1512481; + 1 623 1512728-1512931; 1513310-1513504; 1514282-1514491; 1514642-1514861; 1515345-1515373 CM008342.1-snap.4060 14068276-14068818; 14069053-14069265; 14069351-14069461; + 1 650 14069709-14070491 CM008342.1-snap.4063 14080849-14081390; 14081696-14081906; 14082004-14082114; + 1 023 14082365-14082523 CM008343.1-snap.1532 7100890-7100952; 7101137-7101535; 7101690-7101854; + 1 185 7101930-7102196; 7102266-7102487; 7102713-7102781 CM008343.1-snap.3551 12195237-12195299; 12195510-12195908; 12196093-12196257; + 1 134 12196351-12196617; 12196701-12196940 CM008344.1-snap.5492 22226248-22226507; 22227548-22228337 + 1 050 1) 基因名称由以“-”分隔的染色体序列文件名和基因编号2部分组成，例如CM008332.1-snap.12826表示NCBI中‘泰中6号’基因组[9]的染色体序列文件CM008332.1.fasta(第2号染色体)上根据snap预测[10]的编号为snap.12826的基因；基因由外显子组成，表中外显子的位置区间均以正义链坐标为准，链为“+”的基因取正义链的序列，链为“−”的基因取正义链反向互补的序列，外显子排列顺序按照其在基因中出现的顺序，拼接外显子即可得到基因全长序列
1) The gene name consists of the name of chromosome sequence file and the gene number that are separated by the symbol “-”, for example, the CM008332.1-snap.12826 indicates the gene numbered snap.12826, which is predicted with snap[10] in the chromosome sequence file CM008332.1.fasta (chromosome No. 2) of the ‘Taizhong 6’ genome[9] in NCBI; The genes are made up of exons, in the table, the positional interval of the exon is set according to the sense strand, the sequence of gene with strand “+” is taken from the sense strand and the sequence of gene with the strand “−” is taken from the reverse compliment of the sense strand, the exons are arranged corresponding to the sequential position within the gene, the full-length gene can be obtained via merging the exons
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表 2 甘薯β−淀粉酶保守基序氨基酸组成特征1)
Table 2 Amino acid composition of the conservative motifs of the Ipomoea batatas β-amylase
基序 Motif 氨基酸组成 Amino acid composition 结构域 Structural domain Motif 1 VDVWWGLVEKDSPREYNWAGYSELLQLAKKHGLKVQA VMSFHQCGGNVGD Glyco_hydro_14 Motif 2 GIHWWYGTRSHAAELTAGYYNTRGRDGYLPIARMLARHGAT LNFTCLEMR Glyco_hydro_14 Motif 3 IPLPRWVLEEGDKNPDIFYTDRAGRRNYEYLSLGVDNQPLFKGRTPLQMY Glyco_hydro_14 Motif 4 WVFPGIGEFQCYDKYMVASWKGAAEAAGHPEWGMPGPTDAG Glyco_hydro_14 Motif 5 TEFFRENGTYNTDYGKFFLTWYSQMLIIHGDRILQEANKVF Glyco_hydro_14 Motif 6 FLLGGTIVDIQVGM GPAGELRYPSYPETQ — Motif 7 VKMDHTMNRKKAMEVSLQALKSAGVEGVM — Motif 8 IPKMMSRSRGVPVFVMLPLD — Motif 9 MCAFTYLRMNPELFEARNWIQFVGFVKKMKEGEQRREC SCOP domain d1byb_ Motif 10 DHEQPQHAQCAPEKLVWQVLLATWEARVPLAGENALPRYD Glyco_hydro_14 1) 下划线表示与图1对应的保守区域部分；“—”表示未匹配到相关信息
1) The underlines indicate the conservative regions corresponding to Fig. 1; “—” indicates no relevant information is matched
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表 3 β−淀粉酶蛋白数据库查询编号
Table 3 The database query numbers of β-amylase proteins
物种
Species蛋白名称
Protein name数据库查询编号
Database query number物种
Species蛋白名称
Protein name数据库查询编号
Database query number水稻
Oryza sativaOsBAM1 LOC_Os02g03690.1 拟南芥
Arabidopsis thalianaAtBAM1 AT3G23920.1 OsBAM2 LOC_Os07g35940.1 AtBAM2 AT4G00490.1 OsBAM3 LOC_Os03g04770.1 AtBAM3 AT4G17090.1 OsBAM4 LOC_Os07g47120.1 AtBAM4 AT5G55700.1 OsBAM5 LOC_Os03g22790.1 AtBAM5 AT4G15210.1 OsBAM6 LOC_Os09g39570.1 AtBAM6 AT2G32290.1 OsBAM7 LOC_Os10g32810.1 AtBAM7 AT2G45880.1 OsBAM8 LOC_Os01g13550.1 AtBAM8 AT5G45300.1 OsBAM9 LOC_Os10g41550.1 AtBAM9 AT5G18670.1 OsBAM10 LOC_Os07g35880.1 马铃薯
Solanum tuberosumStBAM1 PGSC0003DMP400002800 玉米
Zea maysZmBAM1 NP_001148159.2 StBAM3 PGSC0003DMP400035625 ZmBAM2 NP_001151271.2 StBAM4 PGSC0003DMP400021443 ZmBAM3 XP_008658465.1 StBAM5 PGSC0003DMP400045472 ZmBAM4 NP_001168436.1 StBAM7 PGSC0003DMP400000367 ZmBAM5 NP_001130896.1 StBAM8 PGSC0003DMP400041754 ZmBAM6 AQK60892.1 StBAM9 PGSC0003DMP400018848 ZmBAM7 NP_001354441.1 番茄
Solanum lycopersicumSlBAM1 A0A3Q7I9I2 ZmBAM8 NP_001105496.2 SlBAM2 A0A3Q7EKY0 ZmBAM9 NP_001170007.1 SlBAM3 A0A3Q7HEM6 ZmBAM10 NP_001337631.1 SlBAM4 A0A3Q7IDQ8 ZmBAM11 NP_001132696.1 SlBAM5 A0A3Q7IEI0 ZmBAM12 XP_035818275.1 SlBAM6 A0A3Q7HUA2 大豆
Glycine maxGmBAM1 P10538.3 SlBAM7 A0A3Q7HV50 GmBAM2 CAI39245.1 大麦
Hordeum vulgareHvBAM1 CAC16789.1 GmBAM3 CAI39244.1 HvBAM2 AAX37357.1
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