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作物学报 ›› 2018, Vol. 44 ›› Issue (11): 1661-1672.doi: 10.3724/SP.J.1006.2018.01661

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

阻断授粉诱导玉米叶片提前衰老的转录组分析

吴连成,李沛,田磊,王顺喜,李明娜,王宇宇,王赛,陈彦惠()   

  1. 河南农业大学农学院 / 河南粮食作物协同创新中心 / 小麦玉米作物学国家重点实验室, 河南郑州 450046
  • 收稿日期:2018-01-02 接受日期:2018-07-20 出版日期:2018-11-12 网络出版日期:2018-07-30
  • 通讯作者: 陈彦惠
  • 基金资助:
    本研究由国家重点研发计划项目(2016YFD0101205-3);河南省科技厅基础前沿项目(142300413218)

Transcriptome Analysis of Premature Senescence Induced by Pollination-prevention in Maize

Lian-Cheng WU,Pei LI,Lei TIAN,Shun-Xi WANG,Ming-Na LI,Yu-Yu WANG,Sai WANG,Yan-Hui CHEN()   

  1. College of Agronomy, Henan Agricultural University / Collaborative Innovation Center of Henan Grain Crops / National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450046, Henan, China
  • Received:2018-01-02 Accepted:2018-07-20 Published:2018-11-12 Published online:2018-07-30
  • Contact: Yan-Hui CHEN
  • Supported by:
    This study was supported by the National Key Research and Development Program of China(2016YFD0101205-3);the Basic Frontier Project of Henan Science and Technology Department(142300413218)

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摘要：

衰老发生在玉米生长发育的最后阶段, 叶片适时启动衰老对玉米最终产量的形成具有至关重要的作用。本研究以玉米自交系豫816为试材, 采用RNA-seq技术分析阻断授粉诱导玉米叶片提前衰老的分子机制。与正常授粉植株相比, 阻断授粉诱导条件下的植株在吐丝后27 d, 叶片全部变黄并枯萎。吐丝后24 d, 授粉植株与同期非授粉植株叶片间的叶绿素含量差异达到极显著水平。差异表达基因(DEG)分析结果显示, 吐丝后10 d的授粉处理组与同期非授粉处理组比较存在173个DEG; 吐丝后24 d的授粉处理组与同期非授粉处理组比较存在835个DEG。吐丝后24 d的授粉处理组与吐丝后10 d的授粉处理组比较存在1381个DEG; 吐丝后24 d的非授粉处理组与吐丝后10 d的非授粉处理组比较存在1591个DEG。GO功能富集分析发现, 吐丝后10 d, 授粉处理组和非授粉处理组间DEG的功能主要富集在刺激响应和代谢进程; 吐丝后24 d, 授粉处理与非授粉处理间DEG的功能主要富集在光合作用进程。Pathway富集分析结果显示, 吐丝后10 d, 授粉处理与非授粉处理间的DEG主要参与RNA降解、光合作用、木质素合成、转录调控、糖转运代谢路径; 吐丝后24 d, 授粉处理与非授粉处理间的DEG主要参与信号传导、激素代谢和光合作用路径。阻断授粉诱导处理使豫816植株体内碳水化合物代谢和光合作用受到影响, 造成玉米衰老反应的启动和衰老速率显著提前和加快。

关键词: 玉米, 阻断授粉, 早衰, 转录组

Abstract:

Senescence occurs in the last stage of maize growth and development. Timely started leaf aging has a crucial role on the formation of maize final yield. Maize inbred line Yu 816 was used to explore the molecular mechanism of early senescence induced under pollination-prevention by transcriptome analysis. Compared with the normal pollination plants, the leaves of non-pollination plants turned yellow and withered at 27 days after silking (DAS). Leaf chlorophyll content difference between pollination and non-pollination plants reached extremely significant level at 24 DAS. RNA-seq assay revealed there were 173 and 835 differentially expressed genes (DEGs) between pollinated and non-pollinated treatments at 10 DAS and 24 DAS, respectively. There were 1381 DEGs in pollination treatment group and 1591 DEGs in non-pollination treatment group between 10 DAS and 24 DAS. GO analysis showed that DEG functions between pollination and non-pollination treatments were mostly enriched in stimulus response and metabolic process at 10 DAS, whereas mainly in photosynthesis process at 24 DAS. Furthermore, pathway enrichment analysis showed that DEGs between pollinated and non-pollinated treatments were mainly involved in the metabolic pathways such as RNA degradation, photosynthesis, lignin synthesis, transcription regulation and sugar transport at 10 DAS, while primarily in the processes of signaling, hormone metabolism, photosynthesis at 24 DAS. Carbohydrate metabolism and photosynthesis processes affected by pollination-prevention result in the senescence onset and the significantly fast aging ahead of schedule in Yu 816 plants.

Key words: maize, pollination-prevention, premature senescence, transcriptome

表1

RT-qPCR分析中用到的引物"

基因编号
Gene ID		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
GRMZM2G033493 AATGCAACGGAGCCAACAAT TTTGTGACAGCTTCGTTCGG
GRMZM2G035243 CCAGCCATCCGTCTATCCAT TCTAATCTTGCAGCGCGAAC
GRMZM2G109070 GTGTACTACGAGAGGTCCGG AAAGCCCCAAAACGCATCTT
GRMZM2G117198 GGACACATGTTCGGGTATGC ATTGGTCACTGTCTCGTCGT
GRMZM2G339563 ATCGTTCTTCAAGGCCAGGA CATCTCGCGCTTTGAAAGGA
GRMZM5G801627 TGTGCAGGCGACCATGTATA CATCAACTCAAGACGCCGTT
GRMZM2G088053 AGAGTGAGGCCCAAGATGAC CTCAGCCTCTCCATCCTCAG
GRMZM2G109627 CGAGGATAACTGCAACGGTG GTCGTGCAGCTGATGAGAAG
GRMZM2G062129 AGCAAGTCTGATGGCTCACT AGCCAACCCTTGACTAGCAT
GRMZM2G064962 CGAAACACCACGATCCAAGG ATGTAGACTGCCTCCCACAC

图1

授粉和非授粉条件下豫816表型变化图中DAS表示吐丝后天数; POL表示授粉; Non-POL表示非授粉。"

图2

非授粉和授粉植株穗位叶叶绿素含量变化**表示非授粉(Non-POL)和授粉(POL)植株穗位叶叶绿素含量之间差异极显著(P < 0.01)。"

表2

样品reads数分布情况"

样本
Sample		 原始数据
Raw data count		 过滤后的
reads数量
Count
after filter		 过滤后
reads占比
Reads keep rate (%)		 比对上的reads数量Mapped reads 比对上的
reads占比
Mapped reads
rate (%)		 在参考序列上有
唯一比对位置的
reads数量
Unique mapped reads		 有唯一比对位置的reads占比
Unique mapped reads (%)
10DASN_r1 7442024 7291875 97.98 6731287 92.31 5480898 81.42
10DASN_r2 9376032 9181843 97.93 8479906 92.36 7106564 83.80
10DASY_r1 7941949 7777455 97.93 7134198 91.73 5833995 81.78
10DASY_r2 8834814 8644115 97.84 7960889 92.10 6607957 83.01
24DASN_r1 9382351 9163732 97.67 8367413 91.31 7079381 84.61
24DASN_r2 8201533 8054678 98.21 7433771 92.29 6190232 83.27
24DASY_r1 8599542 8432296 98.06 7728019 91.65 6398312 82.79
24DASY_r2 9545411 9339804 97.85 8557539 91.62 7181523 83.92
24DASY_r3 8206919 8027609 97.82 7381500 91.95 6095486 82.58

图3

生物学重复间主成分分析表中N表示非授粉处理; Y表示授粉处理; r表示生物学重复。"

图4

差异表达基因RT-qPCR验证"

图5

差异表达基因的上调下调数目“10N-10Y”代表吐丝后10 d授粉处理组与非授粉处理组, “24N-24Y”代表吐丝后24 d授粉处理组与非授粉处理组, “10Y-24Y”代表授粉处理组吐丝后24 d与授粉植株吐丝后10 d, “10N-24N”代表非授粉处理组吐丝后24 d与非授粉处理吐丝后10 d。"

图6

差异表达基因Venn图维恩图中非重叠区的数字表示在一个成对比较中特有的差异表达基因数目, 重叠区的数字表示在不同成对比较中共有的差异表达基因数目。"

图7

差异表达基因的GO功能分析"

图8

差异表达基因的Pathway富集分析"

表3

吐丝后10 d参与阻断授粉诱导玉米提前衰老相关基因"

玉米基因编号
Maize gene ID		 差异倍数
FC (10N/10Y)		 基因功能
Gene function
GRMZM2G127846 9.51 Exonuclease family protein
GRMZM2G009223 6.56 Glucose-6-phosphate/phosphate translocator 2 (GPT2)
GRMZM2G339562 6.17 Response to low sulfur 4 (LSU4)
GRMZM2G087254 5.78 APS reductase 3 (APR3)
GRMZM2G071630 3.90 Glyceraldehyde-3-phosphate dehydrogenase C2
GRMZM2G345700 3.83 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein
GRMZM2G139874 3.66 Cinnamate-4-hydroxylase (C4H)
GRMZM2G000264 3.33 H(+)-ATPase 11
GRMZM2G079613 3.23 Tetratricopeptide repeat-like superfamily protein
GRMZM2G144346 0.35 B-box type zinc finger protein with CCT domain
GRMZM2G123896 0.33 Dormancy/auxin associated family protein
GRMZM2G051151 0.31 Oxidative stress 3 (OXS3)
GRMZM2G106792 0.30 NDR1/HIN1-like 2
GRMZM5G801949 0.28 Sugar transporter 4 (STP4)
玉米基因编号
Maize gene ID		 差异倍数
FC (10N/10Y)		 基因功能
Gene function
GRMZM2G068510 0.24 S-adenosyl-L-methionine-dependent methyltransferases superfamily protein
GRMZM2G172214 0.22 CBS domain containing membrane protein
AC208201.3_FG002 0.20 Protein phosphatase 2C family protein
GRMZM2G412601 0.19 SOS3-interacting protein 3 (SIP3)
GRMZM2G088819 0.16 Calcium-binding EF-hand family protein
GRMZM2G084958 0.15 Protochlorophyllide oxidoreductase A (PORA)
GRMZM2G478553 0.15 RING/U-box superfamily protein (ATL3)
GRMZM2G142802 0.14 Aluminium induced protein
GRMZM2G149024 0.12 Galacturonosyltransferase-like 2
GRMZM2G177050 0.11 SOS3-interacting protein 4 (SIP4)
GRMZM2G131055 0.08 Glycosyltransferase family 61 protein

表4

吐丝后24 d豫816穗位叶中碳水化合物代谢基因"

玉米基因编号
Maize gene ID		 差异倍数
FC (24N/24Y)		 基因功能
Gene function
GRMZM2G001304 0.19 Trehalose-6-phosphate synthase (TPS)
GRMZM2G068943 0.22 Trehalose-6-phosphate synthase (TPS)
GRMZM2G112830 4.55 Trehalose-phosphatase (TPP)
GRMZM2G099860 4.04 Trehalose-phosphatase (TPP)
GRMZM2G140614 2.75 Glucose-6-phosphate isomerase
GRMZM2G076075 6.43 Glucose-6-phosphate isomerase
GRMZM2G106213 40.55 ADP glucose pyrophosphorylase (AGPase)
GRMZM2G348551 6.92 Starch synthase 2 (SS2)
GRMZM2G089136 0.07 Phosphoglycerate kinase
GRMZM2G104632 2.86 Glyceraldehyde 3-phosphate dehydrogenase (GAP-DH)
GRMZM2G345493 0.09 Fructose-bisphosphate aldolase
GRMZM2G155253 0.05 Fructose-bisphosphate aldolase
玉米基因编号
Maize gene ID		 差异倍数
FC (24N/24Y)		 基因功能
Gene function
GRMZM2G089365 8.92 Fructose-bisphosphate aldolase
GRMZM2G046284 0.16 Fructose-bisphosphate aldolase
GRMZM5G836250 0.11 Fructose-1,6-bisphosphatase
GRMZM2G306732 0.10 Fructose-1,6-bisphosphatase
GRMZM5G875238 0.16 Sucrose phosphate synthase
GRMZM2G466780 0.20 Fructokinase-like 1 (FLN1)
GRMZM2G103843 0.28 Fructokinase-like 2 (FLN2)
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