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猴面花miRNA及靶基因的生物信息学预测



全 文 :书 [收稿日期] 2014-06-20;2015-01-07修回
 [基金项目] 海南省重大科技项目子课题“热带生物种质与基因资源研究”(ZDZX2013023-1)
 [作者简介] 李崇奇(1979-),男,副教授,在读博士,从事分子生物学研究。E-mail:cqlisxlf@163.com
 *通讯作者:周 鹏(1963-),男,研究员,从事作物遗传育种和农业生物技术研究。E-mail:zhp6301@126.com
[文章编号]1001-3601(2015)01-0002-0008-05
猴面花miRNA及靶基因的生物信息学预测
李崇奇1,2,3,沈文涛2,言 普2,黎小瑛2,周 鹏1,2
(1.海南大学 农学院,海南 海口570228;2.中国热带农业科学院热带生物技术研究所 分析测试中心,
海南 海口571101;3.海南医学院 生物化学与分子生物学教研室,海南 海口571199)
  [摘 要]以猴面花基因组序列为试材,通过psRobot网站进行茎环结构预测,应用blast-2.2.27+软件
与rfam数据库和pfam数据库比对后去除非miRNA序列,应用bioedit分析miRNA序列及前体序列的碱
基组成特点。结果表明:猴面花基因组中共发现105条成熟 miRNA序列,分别属于49个不同 miRNA家
族;miRNA序列和其侧翼序列都存在碱基偏倚现象,miRNA5′端第1碱基和第19碱基尿嘧啶和胞嘧啶出
现的频率分别为67.6%和49.5%;93个miRNA预测到了靶基因,其中有64个靶向转录因子。
[关键词]猴面花;miRNA;基因组序列;转录因子
[中图分类号]Q74 [文献标识码]A
Bioinformatic Prediction of microRNA and Their Target Gene in Mimulus gutatus
LI Chongqi 1,2,3,SHEN Wentao2,YAN Pu2,LI Xiaoying2,ZHOU Peng1,2*
(1.College of Agronomy,Hainan University,Haikou,Hainan 570228;2.Institute of Tropical Bioscience and
Biotechnology/Analysis &Testing Center,Chinese Academy of Tropical Agricultural Sciences,Haikou,Hainan
571101;3.Department of Biochemistry and Molecular Biology,Hainan Medical College,Haikou,Hainan
571199,China)
  Abstract:Taking genome sequence of M.gutatus as material,prediction of stem loop structure of pre-
miRNAs was run on the web-based software psRobot,then non-miRNA sequences were removed by
BLAST search against rfam and pfam database.The characteristics of base composition was analyzed by
bioedit software in miRNA sequences and their precursors.The result showed that there are 105mature
sequences in M.gutatus which belong to 49different miRNA familes.The base bias phenomenon has been
found in miRNA sequence and their flanking sequences.The frequency of uracil and cytosine is respectively
67.6%and 49.5%in the first base and nineteenth base from miRNA 5′end.The target genes has been
found in 93miRNAs,in which 64miRNAs regulate transcription factors.
Key words:Mimulus gutatus;miRNA;genome sequence;transcription factor
  猴面花(Mimulus gutatus)为玄参科沟酸浆属
草本植物,原产于北美西部,现在世界范围内被广泛
栽培。猴面花喜湿润、光照充足且冷凉的环境[1],生
长适温为18~21℃,可用于公园、小区的花坛、花
台、花境栽培观赏,也是庭院及居室栽培的优良材
料[2]。猴面花具基因组小、代时短、高繁殖力、自交
亲和性和易栽培等特性,因而常被应用于植物对土
壤污染适应[3]、交配系进化[4]和近交衰退[5]等生态
和进化遗传学研究领域的模式生物[6]。目前,对于
猴面花的miRNA识别及调控机制的研究非常少,
仅Barozai等[7]应用EST序列识别了28条猴面花
miRNA 序 列 和 6 条 刘 易 斯 猴 面 花 (Mimulus
lewisii)miRNA序列。而猴面花基因组测序的完
成,可在全基因组范围内识别猴面花 miRNA基因
提供了可能。因此,拟应用psRobot[8]在线分析工
具,在猴面花全基因组范围内识别其 miRNA序列,
预测miRNA靶基因,为探讨猴面花独特的生态特
点和进一步开展猴面花遗传品质改良工作奠定理论
基础。
1 材料与方法
1.1 试验材料
从miRBase(http://www.mirbase.org/)数据
库中下载所有植物的成熟miRNA序列,共计5 940
条;猴面花全基因组序列已经被psRobot网站预置
在其数据库中,直接下载。此外,非编码RNA数据
库和蛋白质数据库则分别从rfam(http://rfam.
sanger.ac.uk/)网站和pfam(http://pfam.sanger.
ac.uk/)网站下载。
1.2 试验方法
将所有植物miRNA序列去除重复序列后上传
到psRobot网站(http://omicslab.genetics.ac.cn/
psRobot/index.php)进行茎环结构预测,选择松弛
参数模式,将预测到的茎环结构序列去除重复序列
后,应用blast-2.2.27+软件中的blastn程序与
rfam数据库进行比对,除去除 miRNA外的非编码
RNA序列;用blastx程序与pfam数据库进行比对
去除蛋白质序列,将evalue参数设置为1e-6,其他
 贵州农业科学 2015,43(1):8~12
 Guizhou Agricultural Sciences
参数默认。然后计算茎环结构序列AU含量,并参照
Zhang等[9]的方法计算 MFEI值,筛选出大于0.85的
即为预测到的miRNA的前体序列,同时可以得到其
对应的成熟miRNA序列。应用bioedit统计miRNA
及其前体的序列长度,然后统计miRNA成熟序列每
个位点的碱基组成,对其碱基偏倚进行分析。分别在
前体序列中提取成熟miRNA 5′端和3′端10bp碱基
侧翼序列,然后应用bioedit软件分析每个位点的碱
基组成情况。将预测到的成熟 miRNA序列以fasta
格式上传到psRobot网站(http://omicslab.genetics.
ac.cn/psRobot/index.php),应用靶基因在线工具进
行预测,参数选择严格模式。
2 结果与分析
2.1 猴面花的miRNA预测
将5 940条植物miRNA序列去除重复序列后,
共获得3 228条序列作为候选的 miRNA序列。将
候选序列提交到psRobot网站分析后共发现具有茎
环结构的序列304条,去除重复序列后得131条序
列。与rfam数据库和pfam数据库比对后发现1条
tRNA序列,没有发现除 miRNA外的非编码序列
和蛋白质序列。候选前体序列的最小自由能指数分
析发现105条大于0.85的序列,即为 miRNA的前
体序列,相对应的 miRNA 同源序列即为成熟的
miRNA序列,miRNA前体序列和成熟序列的参数
详见表1。分析发现,105条成熟 miRNA序列属于
49个不同 miRNA家族,miR166家族成员最多,有
8个成员。预测到的所有 miRNA前体序列都具有
典型的茎环结构,AU 含量为46.5%~71.0%,其
最小自由能均为负值,最高的为 mgu-miR395h,其
自由能为-33.1kcal/mol。
表1 预测的105个猴面花的miRNA及其前体的序列相关参数
Table 1 105miRNAs identified in M.gutatus and the characteristics of their precursors
序号
No.
名称
Name
miRNA序列
miRNA
sequences
miRNA
序列长度
ML
前体序
列长度
PL
前体序列位置
LPS
最小自由能/
(kcal/mol)
MFE
A+
U/%
最小自由
能指数
MFEI
1 mgu-miR390a AAGCUCAGGAGGGAUAGCACC  21  101 scaffold_101:-:196451-196551 -44.9  58.4  1.07
2 mgu-miR390b AAGCUCAGGAGGGAUAGCGCC  21  111 scaffold_1:+:792654-792764 -43.1  55.0  0.86
3 mgu-miR156a ACAGAAGAUAGAGAGCACAG  20  90 scaffold_11:-:2526010-2526099 -44.1  63.3  1.34
4 mgu-miR172a AGAAUCUUGAUGAUGCUGCA  20  140 scaffold_2:+:1320468-1320607 -55.1  54.3  0.86
5 mgu-miR172b AGAAUCUUGAUGAUGCUGCAG  21  121 scaffold_8:+:2190808-2190928 -57.6  57.9  1.13
6 mgu-miR172c AGAAUCUUGAUGAUGCUGCAU  21  141 scaffold_2:+:1320468-1320608 -55.1  54.6  0.86
7 mgu-miR171a AGAUUGAGCCGCGCCAAUAUC  21  101 scaffold_213:-:332936-333036 -40.1  55.4  0.89
8 mgu-miR169a AGCCAAGGAUGACUUGCCGG  20  70 scaffold_17:-:2311583-2311652 -37.2  52.9  1.13
9 mgu-miR390c AGCUCAGGAGGGAUAGCGCC  20  110 scaffold_1:+:792655-792764 -42.6  54.5  0.85
10 mgu-miR395e AUGAAGUGUUUGGGGGAACUC  21  91 scaffold_37:-:293339-293429 -44.5  58.2  1.17
11 mgu-miR169b CAGCCAAGGAUGACUUGCC  19  79 scaffold_17:-:2311575-2311653 -42.9  53.2  1.16
12 mgu-miR169c CAGCCAAGGAUGACUUGCCGA  21  111 scaffold_34:+:1555256-1555366 -48.3  62.2  1.15
13 mgu-miR169d CAGCCAAGGAUGACUUGCCGG  21  81 scaffold_17:-:2311573-2311653 -44.1  53.1  1.16
14 mgu-miR168a CCCGCCUUGCAUCAACUGAAU  21  191 scaffold_181:+:112694-112884 -91.5  48.7  0.93
15 mgu-miR171b CGAUGUUGGUGAGGUUCAAUC  21  101 scaffold_213:-:332939-333039 -40.0  56.4  0.91
16 mgu-miR390d CGCUAUCCAUCCUGAGUUUC  20  110 scaffold_1:+:792653-792762 -43.9  55.5  0.90
17 mgu-miR390e CGCUAUCCAUCCUGAGUUUCA  21  111 scaffold_1:+:792653-792763 -44.0  55.0  0.88
18 mgu-miR166a CGGACCAGGCUUCAUUCCCC  20  110 scaffold_1:+:3090170-3090279 -52.5  60.9  1.22
19 mgu-miR166b CUCGGACCAGGCUUCAUUCCC  21  91 scaffold_255:+:150395-150485 -48.6  52.7  1.13
20 mgu-miR395f CUGAAGUGUUUGGGGGAACUC  21  101 scaffold_37:+:294351-294451 -36.9  59.4  0.90
21 mgu-miR395g  CUGAAGUGUUUGGGGGAACUCC  22  102 scaffold_37:+:312003-312104 -48.4  49.0  0.93
22 mgu-miR156b CUGACAGAAGAUAGAGAGCAC  21  101 scaffold_11:-:2526002-2526102 -50.1  61.4  1.28
23 mgu-miR319a CUUGGACUGAAGGGAGCUCC  20  200 scaffold_1:-:1064397-1064596 -85.6  55.0  0.95
24 mgu-miR319b CUUGGACUGAAGGGAGCUCCC  21  201 scaffold_1:-:1064396-1064596 -85.6  55.2  0.95
25 mgu-miR172d GAAUCUUGAUGAUGCUGCAU  20  140 scaffold_2:+:1320469-1320608 -55.1  54.3  0.86
26 mgu-miR160a GCCUGGCUCCCUGUAUGCCAU  21  101 scaffold_16:-:767280-767380 -50.4  47.5  0.95
27 mgu-miR160b GCGUAUGAGGAGCCAAGCAUA  21  101 scaffold_16:-:767279-767379 -50.4  46.5  0.93
28 mgu-miR156c GCUCACUUCUCUCUCUGUCAGC  22  102 scaffold_273:-:90172-90273 -52.0  58.8  1.24
29 mgu-miR157a GCUCUCUAUGCUUCUGUCAUC  21  111 scaffold_110:-:236645-236755 -49.7  61.3  1.16
30 mgu-miR172e GGAGCAUCAUCAAGAUUCACA  21  121 scaffold_8:+:2190808-2190928 -57.6  57.9  1.13
31 mgu-miR169e GGCAAGUUGUUCUUGGCUACA  21  121 scaffold_34:+:1555249-1555369 -55.5  62.0  1.21
32 mgu-miR396a GUUCAAUAAAGCUGUGGGAA  20  120 scaffold_9:+:900770-900889 -49.0  55.0  0.91
33 mgu-miR396b GUUCAAUAAAGCUGUGGGAAG  21  121 scaffold_9:+:900770-900890 -49.4  55.4  0.91
34 mgu-miR156d GUUGACAGAAGAGAGUGAGCAC  22  112 scaffold_50:+:1450149-1450260 -51.9  60.7  1.18
35 mgu-miR211 UAAUCUGCAUCCUGAGGUUUG  21  101 scaffold_13:-:1353788-1353888 -45.1  56.4  1.03
36 mgu-miR169f UAGCCAAGGAUGACUUGCCGG  21  121 scaffold_233:-:24686-24806 -48.1  62.0  1.05
37 mgu-miR169g  UAGCCAAGGAUGACUUGCCU  20  90 scaffold_115:+:226945-227034 -39.3  56.7  1.01
38 mgu-miR169h UAGCCAAGGAUGACUUGCCUA  21  91 scaffold_115:+:226945-227035 -39.3  57.1  1.01
39 mgu-miR393a UCCAAAGGGAUCGCAUUGAUC  21  111 scaffold_51:-:775389-775499 -47.9  62.2  1.14
40 mgu-miR393b UCCAAAGGGAUCGCAUUGAUCU  22  112 scaffold_51:-:775388-775499 -47.9  61.6  1.11
41 mgu-miR396c UCCACAGCUUUCUUGAACUG  20  110 scaffold_213:+:309675-309784 -45.0  56.4  0.94
·9·
 李崇奇 等 猴面花miRNA及靶基因的生物信息学预测
 LI Chongqi et al Bioinformatic Prediction of microRNA and Their Target Gene in Mimulus gutatus
 续表1
42 mgu-miR396d UCCCACAGCUUUAUUGAACUG  21  111 scaffold_9:-:900778-900888 -44.5  56.8  0.93
43 mgu-miR168b UCGCUUGGUGCAGGUCGGG  19  199 scaffold_181:+:112698-112896 -92.9  48.2  0.90
44 mgu-miR168c UCGCUUGGUGCAGGUCGGGA  20  200 scaffold_181:+:112698-112897 -93.7  48.0  0.90
45 mgu-miR168d UCGCUUGGUGCAGGUCGGGAA  21  201 scaffold_181:+:112698-112898 -93.7  47.8  0.89
46 mgu-miR166c UCGGACCAGGCUUCAUUCC  19  109 scaffold_1:+:3090169-3090277 -52.5  61.5  1.25
47 mgu-miR166d UCGGACCAGGCUUCAUUCCC  20  110 scaffold_1:+:3090169-3090278 -52.5  60.9  1.22
48 mgu-miR166e UCGGACCAGGCUUCAUUCCCC  21  111 scaffold_1:+:3090169-3090279 -52.5  61.3  1.22
49 mgu-miR166f UCGGACCAGGCUUCAUUCCCCC  22  152 scaffold_104:+:474084-474235 -60.5  62.5  1.06
50 mgu-miR166g  UCGGACCAGGCUUCAUUCCU  20  120 scaffold_17:-:2195286-2195405 -50.6  60.0  1.05
51 mgu-miR166h UCGGACCAGGCUUCAUUCCUC  21  121 scaffold_17:-:2195285-2195405 -50.6  60.3  1.05
52 mgu-miR166i UCUCGGACCAGGCUUCAUUC  20  120 scaffold_17:-:2195288-2195407 -50.5  60.8  1.07
53 mgu-miR166j  UCUCGGACCAGGCUUCAUUCC  21  121 scaffold_17:-:2195287-2195407 -50.6  60.3  1.05
54 mgu-miR828 UCUUGCUCAAAUGAGUAUUCCA  22  162 scaffold_8:-:966288-966449 -55.2  71.0  1.17
55 mgu-miR167a UGAAGCUGCCAGCAUGAUCU  20  170 scaffold_1:+:3292699-3292868 -66.7  67.1  1.19
56 mgu-miR167b UGAAGCUGCCAGCAUGAUCUA  21  171 scaffold_1:+:3292699-3292869 -66.7  66.7  1.17
57 mgu-miR167c UGAAGCUGCCAGCAUGAUCUAA  22  92 scaffold_130:-:136075-136166 -43.0  59.8  1.16
58 mgu-miR167d UGAAGCUGCCAGCAUGAUCUC  21  91 scaffold_1:+:3295386-3295476 -41.5  53.8  0.99
59 mgu-miR167e UGAAGCUGCCAGCAUGAUCUG  21  221 scaffold_115:+:249406-249626 -60.6  68.8  0.88
60 mgu-miR167f UGAAGCUGCCAGCAUGAUCUGG  22  222 scaffold_115:+:249406-249627 -60.6  68.9  0.88
61 mgu-miR395h UGAAGUGUUUGGGGGAACUC  20  90 scaffold_37:+:294362-294451 -33.1  60.0  0.92
62 mgu-miR395i UGAAGUGUUUGGGGGAACUUU  21  101 scaffold_37:+:308608-308708 -53.1  50.5  1.06
63 mgu-miR156e UGACAGAAGAGAGAGAGCAC  20  100 scaffold_108:-:335293-335392 -42.0  53.0  0.89
64 mgu-miR156f UGACAGAAGAGAGAGAGCACA  21  101 scaffold_108:-:335292-335392 -42.0  52.5  0.88
65 mgu-miR156g  UGACAGAAGAGAGUGAGCAC  20  110 scaffold_36:-:1711582-1711691 -55.0  50.9  1.02
66 mgu-miR156h UGACAGAAGAGAGUGAGCACA  21  111 scaffold_36:-:1711581-1711691 -55.0  50.5  1.00
67 mgu-miR156i UGACAGAAGAGAGUGAGCAUA  21  101 scaffold_273:+:82259-82359 -46.6  60.4  1.17
68 mgu-miR157b UGACAGAAGAUAGAGAGCAC  20  100 scaffold_11:-:2526002-2526101 -50.1  61.0  1.28
69 mgu-miR171c UGAUUGAGCCGCGCCAAUAU  20  130 scaffold_2:+:4392481-4392610 -46.2  63.1  0.96
70 mgu-miR171d UGAUUGAGCCGCGCCAAUAUC  21  131 scaffold_2:+:4392481-4392611 -46.3  63.4  0.96
71 mgu-miR171e UGAUUGAGCCGCGCCAAUAUCU  22  132 scaffold_2:+:4392481-4392612 -46.3  62.9  0.94
72 mgu-miR171f UGAUUGAGCCGUGCCAAUAU  20  90 scaffold_120:-:404663-404752 -42.4  56.7  1.09
73 mgu-miR171g  UGAUUGAGCCGUGCCAAUAUC  21  91 scaffold_120:-:404662-404752 -42.7  57.1  1.09
74 mgu-miR530a UGCAUUUGCACCUGCACCUC  20  150 scaffold_324:+:67374-67523 -56.7  61.3  0.98
75 mgu-miR530b UGCAUUUGCACCUGCACCUU  20  120 scaffold_2:-:1245956-1246075 -42.9  64.2  1.00
76 mgu-miR399a UGCCAAAGGAGAAUUGCCC  19  89 scaffold_12:-:2334313-2334401 -38.4  56.2  0.98
77 mgu-miR399b UGCCAAAGGAGAAUUGCCCUG  21  91 scaffold_12:-:2334311-2334401 -38.4  56.0  0.96
78 mgu-miR399c UGCCAAAGGAGAGUUGCCCUG  21  111 scaffold_51:-:532535-532645 -38.5  64.9  0.99
79 mgu-miR399d UGCCAAAGGAGAUUUGCCCGG  21  151 scaffold_31:+:995443-995593 -54.2  62.3  0.95
80 mgu-miR160c UGCCUGGCUCCUUGUAUGCCA  21  101 scaffold_17:-:1368915-1369015 -54.0  47.5  1.02
81 mgu-miR164a UGGAGAAGCAGGGCACAUGCC  21  91 scaffold_7:-:456344-456434 -42.1  56.0  1.05
82 mgu-miR164b UGGAGAAGCAGGGCACAUGCU  21  111 scaffold_1:+:432994-433104 -62.3  50.5  1.13
83 mgu-miR164c UGGAGAAGCAGGGCACGUGC  20  110 scaffold_128:+:394816-394925 -48.7  53.6  0.95
84 mgu-miR164d UGGAGAAGCAGGGCACGUGCA  21  111 scaffold_128:+:394816-394926 -50.4  54.1  0.99
85 mgu-miR164e UGGAGAAGGGGAGCACGUGCA  21  111 scaffold_128:-:451679-451789 -53.4  50.5  0.97
86 mgu-miR171h UGUUGGCUCGGCUCACUCAGA  21  121 scaffold_2:+:4392485-4392605 -42.1  62.8  0.94
87 mgu-miR403 UUAGAUUCACGCACAAACUCG  21  131 scaffold_1:-:4616062-4616192 -45.2  64.1  0.96
88 mgu-miR396e UUCAAUAAAGCUGUGGGAAG  20  120 scaffold_9:+:900771-900890 -49.4  55.8  0.93
89 mgu-miR393c UUCCAAAGGGAUCGCAUUGAUC  22  112 scaffold_51:-:775389-775500 -47.9  62.5  1.14
90 mgu-miR396f UUCCACAGCUUUCUUGAACU  20  110 scaffold_213:+:309674-309783 -44.4  55.5  0.91
91 mgu-miR396g  UUCCACAGCUUUCUUGAACUG  21  111 scaffold_213:+:309674-309784 -45.0  55.9  0.92
92 mgu-miR396h UUCCACAGCUUUCUUGAACUU  21  121 scaffold_7:+:2552347-2552467 -51.2  65.3  1.22
93 mgu-miR156j  UUGACAGAAGAGAGAGAGCAC  21  101 scaffold_108:-:335293-335393 -42.0  52.5  0.88
94 mgu-miR156k UUGACAGAAGAGAGAGAGCACA  22  102 scaffold_108:-:335292-335393 -42.0  52.0  0.86
95 mgu-miR156l UUGACAGAAGAGAGUGAGCAC  21  111 scaffold_50:+:1450150-1450260 -51.5  60.4  1.17
96 mgu-miR156m UUGACAGAAGAUAGAGAGC  19  109 scaffold_11:-:2525763-2525871 -54.2  51.4  1.02
97 mgu-miR156n UUGACAGAAGAUAGAGAGCAC  21  111 scaffold_11:-:2525761-2525871 -56.7  51.4  1.05
98 mgu-miR171i UUGAGCCGUGCCAAUAUCAC  20  100 scaffold_4:+:2271877-2271976 -52.0  59.0  1.27
99 mgu-miR171j  UUGAGCCGUGCCAAUAUCACU  21  101 scaffold_4:+:2271877-2271977 -52.0  58.4  1.24
100 mgu-miR319c UUGGACUGAAGGGAGCUCC  19  199 scaffold_1:-:1064397-1064595 -85.6  54.8  0.95
101 mgu-miR319d UUGGACUGAAGGGAGCUCCC  20  200 scaffold_1:-:1064396-1064595 -85.6  55.0  0.95
102 mgu-miR319e UUGGACUGAAGGGAGCUCCCA  21  221 scaffold_103:-:769743-769963 -100.1  55.7  1.02
103 mgu-miR319f UUGGACUGAAGGGAGCUCCCU  21  201 scaffold_1:-:1064395-1064595 -85.6  55.2  0.95
104 mgu-miR394 UUGGCAUUCUGUCCACCUCC  20  160 scaffold_2:+:4397466-4397625 -53.2  65.6  0.97
105 mgu-miR159 UUUGGAUUGAAGGGAGCUCUA  21  201 scaffold_154:-:338061-338261 -81.1  61.2  1.04
 注:ML为 miRNA长度,PL为前体长度,LPS为前体序列的位置,MFE为最小自由能,MFEI为最小自由能指数。
 Note:ML is the length of miRNA sequences,PL is the length of precursor sequences,LPS is the location of precursor sequences,MFE is minimal folding free en-
ergies,MFEI is minimal folding free energy index.
·01·
                                        贵 州 农 业 科 学
                                   Guizhou Agricultural Sciences
表2 猴面花调控转录因子的miRNA
Table 2 The miRNAs which regulate transcription factors
序号
No.
miRNA 靶基因
Targets
转录因子
TF
1 mgu-miR156a mgv1a020728m,mgv1a005903m,mgv1a015969m,mgv1a009499m SPL
2 mgu-miR156b mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a015969m,mgv1a009499m
3 mgu-miR156d mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a011944m,mgv1a009499m,
mgv1a015863m
4 mgu-miR156e,mgu-miR156f,mgu-miR156g  mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a011944m,mgv1a015969m,
mgv1a014682m,mgv1a009499m,mgv1a015863m
5 mgu-miR156h mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a011944m,mgv1a014682m,
mgv1a009499m,mgv1a015863m
6 mgu-miR156i mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a011944m,mgv1a015969m,
mgv1a014682m,mgv1a009499m,mgv1a015863m
7 mgu-miR157b,mgu-miR156j mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a011944m,mgv1a015969m,
mgv1a009499m
8 mgu-miR156k mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a009499m
9 mgu-miR156l mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a011944m,mgv1a009499m,
mgv1a015863m
10 mgu-miR156m mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a015969m,mgv1a014682m,
mgv1a009499m
11 mgu-miR156n mgv1a020120m,mgv1a020728m,mgv1a005903m,mgv1a015969m,mgv1a009499m
12 mgu-miR166a mgv1a001309m,mgv1a001344m,mgv1a001350m HD-ZIPIII
13 mgu-miR166b mgv1a001309m,mgv1a001344m,mgv1a001350m
14 mgu-miR166c mgv1a001309m,mgv1a001317m,mgv1a001322m,mgv1a001340m,mgv1a001344m,
mgv1a001350m
15 mgu-miR166d,mgu-miR166e mgv1a001309m,mgv1a001344m,mgv1a001350m
16 mgu-miR166g mgv1a001309m,mgv1a001317m,mgv1a001322m,mgv1a001340m,mgv1a001344m,
mgv1a001350m
17 mgu-miR166h mgv1a001309m,mgv1a001344m,mgv1a001350m
18 mgu-miR166i,mgu-miR166j mgv1a001309m,mgv1a001317m,mgv1a001322m,mgv1a001340m,mgv1a001344m,
mgv1a001350m
19 mgu-miR156d mgv1a005461m,mgv1a020221m,mgv1a023919m,mgv1a024283m,mgv1a026074m TPR
20 mgu-miR396c mgv1a006773m
21 mgu-miR168b mgv1a006094m
22 mgu-miR156e mgv1a005461m,mgv1a020221m,mgv1a023919m,mgv1a024283m,mgv1a026074m
23 mgu-miR156g mgv1a005461m,mgv1a020221m,mgv1a023407m,mgv1a023919m,mgv1a024283m,
mgv1a026074m
24 mgu-miR156h,mgu-miR156j,mgu-miR156l mgv1a005461m,mgv1a020221m,mgv1a023919m,mgv1a024283m,mgv1a026074m
25 mgu-miR169a,mgu-miR169b mgv1a013110m,mgv1a009941m,mgv1a014546m,mgv1a012967m NF-Y
26 mgu-miR169c mgv1a013110m
27 mgu-miR169d,mgu-miR169f mgv1a013110m,mgv1a009941m,mgv1a014546m
28 mgu-miR169g mgv1a013110m,mgv1a009941m,mgv1a014546m,mgv1a012967m
29 mgu-miR172a,mgu-miR172b,mgu-miR172c,mgu-miR172d mgv1a008461m,mgv1a021246m AP2
30 mgu-miR171a,mgu-miR171c,mgu-miR171d,mgu-miR171e, mgv1a003577m GRAS
mgu-miR171f,mgu-miR171g,mgu-miR171i,mgu-miR171j
31 mgu-miR166c mgv11b022954m NAC
32 mgu-miR164a mgv1a011270m
33 mgu-miR164b mgv1a009356m,mgv1a017707m,mgv1a009375m
34 mgu-miR164c mgv1a009356m,mgv1a017707m,mgv1a009375m,mgv1a010134m,mgv1a011270m,
mgv1a015033m,mgv1a012627m
35 mgu-miR164d mgv1a009356m,mgv1a017707m,mgv1a009375m
36 mgu-miR394 mgv1a011129m
37 mgu-miR319a mgv1a022282m,mgv1a008698m,mgv1a012916m myb
38 mgu-miR319b mgv1a022282m
39 mgu-miR828 mgv1a023545m,mgv1a019124m,mgv1a009700m
40 mgu-miR319c mgv1a022282m,mgv1a008698m,mgv1a012916m,mgv1a022833m
41 mgu-miR319d mgv1a022282m,mgv1a008698m,mgv1a012916m
42 mgu-miR319f mgv1a022282m,mgv1a008698m
43 mgu-miR159 mgv1a022282m
44 mgu-miR160a mgv1a002753m,mgv1a004523m ARF
45 mgu-miR167a mgv1a001422m,mgv1a001449m,mgv1a002020m
46 mgu-miR167d mgv1a001422m,mgv1a001449m
47 mgu-miR160c mgv1a002753m,mgv1a020932m,mgv1a021021m,mgv1a021154m
48 mgu-miR167a,mgu-miR167e mgv1a024870m PLATZ
49 mgu-miR394 mgv1a014053m,mgv1a014202m
50 mgu-miR166c,mgu-miR166g,mgu-miR166i,mgu-miR166j  mgv1a001410m HOX
51 mgu-miR390c mgv1a014878m bHLH
52 mgu-miR396e mgv1a008797m SSTF
53 mgu-miR172d mgv1a003939m WRKY
54 mgu-miR156a mgv1a000377m zinc finger
 注:TF为转录因子,SPL为SPL转录因子家族,HD-ZIPIII为 HD-ZIPIII转录因子家族,TPR为TPR转录因子,NF-Y为核因子,ARF为生长素反应因子家族,HOX为 HOX转录因子家族,
bHLH 为螺旋-环-螺旋转录因子家族,SSTF为DNA特异性结合转录因子。
 Note:TF is transcription factor,SPL is squamosa promoter binding protein-like,HD-ZIPIII is homeobox-leucine zipper family protein/lipid-binding START domain-containing protein,TPR is
Tetratricopeptide repeat(TPR)-like superfamily protein,NF-Y is nuclear factor Y,ARF is auxin response factor,HOX is homeobox gene,bHLH is basic helix-loop-helix DNA-binding superfamily
protein,SSTF is sequence-specific DNA binding transcription factors.
·11·
 李崇奇 等 猴面花miRNA及靶基因的生物信息学预测
 LI Chongqi et al Bioinformatic Prediction of microRNA and Their Target Gene in Mimulus gutatus
2.2 猴面花miRNA和前体的碱基组成特征
猴面花的 miRNA长度为19~22nt,其中,58
个miRNA为21nt,占55.2%。miRNA序列中嘌
呤与嘧啶的比值为1.1。碱基组成分析发现,A、G、
C、U频率最高的位置分别为5′端第10碱基、第8
碱基、第19碱基和第1碱基,频率分别为41.0%、
53.3%、49.5%和67.6%。miRNA 前体长度为
70~222nt,平均123nt,嘌呤与嘧啶的比值为
1.00∶1.08,碱基组成 A∶G∶C∶U 为1.00∶
0.85∶0.78∶1.22,尿嘧啶的比例明显偏高。前体
序列5′端碱基组成分析发现前9个碱基中嘌呤的比
例都明显高于嘧啶,嘧啶出现频率最低的为第1碱
基,为33.3%;最高的第7碱基为45.7%。miRNA
上游10nt侧翼序列碱基 A∶G∶C∶U 为1∶
1.07∶0.57∶1.51,尿嘧啶比例明显偏高,胞嘧啶明
显偏低;而miRNA下游10nt侧翼序列碱基的A∶
G:∶C∶U为1∶0.65∶0.89∶1.15,鸟嘌呤比例
明显偏低。值得注意的是,下游第1碱基中鸟嘌呤
出现频率仅为9.5%。
2.3 猴面花的miRNA靶基因预测
105个miRNA中有93个预测到了靶基因,共
发现290条编码序列受到 miRNA调控,去除不能
被注释的基因后共发现250条靶基因序列。同时发
现靶基因中有61个为转录因子,并且受到总计62
个miRNA的调控(表2)。仅受单个 miRNA调控
的转录因子编码序列有22条,2个 miRNA调控的
有6条,受调控最多的为SPL转录因子家族(squa-
mosa promoter binding protein-like,SPL)的
mgv1a005903m、mgv1a009499m 和 mgv1a020728m
等3条编码序列,均受到14个miRNA的调控。
3 结论与讨论
1)应用生物信息学方法对猴面花全基因组碱
基序列近些分析,识别了49个不同 miRNA家族的
105条保守 miRNA 序列。Barozai等[7]应用 EST
序列进行预测时共发现28条猴面花的 miRNA序
列和6条刘易斯猴面花(Mimulus lewisi)的 miR-
NA序列,其中只有2条序列为保守序列。本研究
中也预测到了2条保守序列,即 mgu-miR395f和
mgu-miR156j,同时发现,在刘易斯猴面花中有单碱
基突变的 mle-MIR156a和 mle-MIR156b是保守
的,分别为 mgu-miR156e和 mgu-miR157b;而本研
究预测到的其余101条保守miRNA序列均为首次
在猴面花中发现。然而与在水稻(Oryza sativa)中
发现的713条、黄豆(Glycine max)中发现的554条
miRNA序列相比(www.mirbase.org),猴面花的
miRNA还有待于进一步的生物信息学挖掘,或通
过基因克隆、芯片技术和小RNA测序等相关实验
技术进行识别。然而如何识别真正的 miRNA 序
列,避免将其他非编码 RNA或干扰 RNA注释为
miRNA,一直是一项困难的工作。Taylor等[10]对
mirBase数据库中的分析发现,32.3%的miRNA序
列缺少充足的证据证明其为真正的 miRNA序列,
同时否定了数据库中四分之三的 miRNA家族。而
psRobot可以自动化识别用户上传的小RNA分子
是否具有茎环结构,同时可以预测预置基因组物种
的miRNA靶基因情况。Wu等[8]应用拟南芥miR-
NA序列进行测试时发现其识别效率达到94%,而
对靶基因的识别效率也达到89%。
2)尿嘧啶在猴面花miRNA 5′端第1碱基出现
的频率高达67.6%,而胞嘧啶尿嘧啶在第19碱基
出现的频率则高达49.5%,这与在拟南芥、水稻、大
豆和亚麻等其他物种中的发现一致[11-13]。对猴面花
miRNA侧翼序列分析发现,上游序列中尿嘧啶比
例明显偏高,而胞嘧啶明显偏低;而下游序列中鸟嘌
呤比例明显偏低,鸟嘌呤在下游第1碱基中出现频
率仅为9.5%。目前研究发现,成熟miRNA序列5′
端第一碱基的种类不同,可能导致其选择不同的
Argonaute蛋白,从而形成不同的RISC复合物[14]。
但是对于 miRNA 初级转录产物在加工及形成
RISC复合物的过程中,除了对miRNA序列本身的
碱基有选择性以外,是否会对 miRNA侧翼序列具
有选择性,还有待于进一步研究。
3)猴面花中93个预测到了靶基因的 miRNA
中,有64个靶向转录因子。同时发现不同的猴面花
转录因子家族主要受不同的 miRNA家族调控,如
SPL转录因子家族主要受到猴面花 miRNA156家
族的调控,HD-ZIPIII转录因子家族受 miRNA166
家 族 调 控,核 因 子 受 miRNA166 家 族 调 控。
Jorgensen和Preston[15]认为,SPL转录因子家族可
能通过调控开花时间基因的表达决定物种是多年生
植物还是一年生植物。同时近年来在植物研究中发
现,miRNA156家族通过调控SPL转录因子家族决
定植物发育时相的转变;miRNA159 通过调控
MYB转录因子家族会导致开花推迟;miRNA164
家族通过调控NAC转录因子家族影响花和根的发
育[16]。然而在猴面花中 miRNA如何通过调控这
些转录因子影响植物的发育,其调控方式和其突变
后产生的表型是否与在其他植物中一致都还需要进
一步研究。
[参 考 文 献]
[1] 张 亚.猴面花“花脸”系列栽培技术[J].中国花卉园
艺,2010(10):37.
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32. (下转第15页)
·21·
                                        贵 州 农 业 科 学
                                   Guizhou Agricultural Sciences
育秧方式采用旱育或两段育秧技术;播种前晒种、强
氯精浸种、稀播匀播,科学肥水管理,充分利用光温
条件,培育多蘖壮秧。
4.2 适时移栽,合理密植
秧龄以38~45d为宜,金优990分蘖力中等,
要协调合理密度来提高产量,随着海拔的升高或土
地肥力降低适当增加密植[4],适宜密度为22.5万~
24 万/hm2,每 穴 插 2 粒 谷 秧,基 本 苗 保 证 在
115万~130万/hm2,有利提高有效穗,以夺高产。
4.3 肥水管理
决定水稻产量的主因子是结实率[5],金优990
生育期短,营养生长期相对短,不利于大穗的形成,
移栽时,大田要重施底肥早施分蘖肥,时时控水,湿
润管理,以提高成穗率,从而获得高产。进入灌浆散
籽期后,田间不宜过早脱水,以免影响分蘖穗弱势籽
粒灌浆,导致其籽粒充实度不够影响产量。
4.4 病虫害防治
根据当地水稻生产病虫害发生特点,做到预防
为主,加强病虫害预测预报工作。秧田期做好稻水
象甲的防治工作,分蘖高峰期重点防治稻纵卷叶和
螟稻飞虱,孕穗期至始穗期主要防治二化螟,抽穗扬
花期至成熟期重点防治稻瘟病,科学管理,确保丰产
安全,达到增收增效。
4.5 制种技术要点
恢复系与不育系花期相遇是杂交水稻制种获得
高产的主要因素,金优990在贵州省内制种宜选择
在热量条件、隔离条件好的低海拔地区实施,一般情
况下,4月上中旬播种,叶差4.8叶,父母本播差期
为18d左右。父母本生育期都较短,宜密植和多本
栽插,每穴3~5粒谷,35万穴左右/hm2。
[参 考 文 献]
[1] 杨昌达,黄宗洪,黄伟秀,等.贵州稻作[M].贵阳:贵
州科技出版社,2010:1-2.
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优302的选育及应用[J].贵州农业科学,2007,26
(9):88-90.
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合奇优894[J].种子,2011,30(8):119-121.
[4] 张家洪,谭美林,王庆伟,等.不同密度与育秧方式对
金优990产量的影响[J].耕作与栽培,2014(1):16-
21..
[5] 张家洪,谭美林,王庆伟,等黔中地区杂交水稻播始历
期与若干经济性状的相关性分析[J].南方农业,2011
(7):3-5.
(责任编辑:姜 萍
櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁櫁

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·51·
 张家洪 等 早熟高产杂交水稻新组合金优990的选育
 ZHANG Jiahong et al Breeding of Jinyou 990,a New Hybrid Rice Combination with Early Maturity and High Yield