基于转录组测序的高羊茅分蘖与株高相关差异表达基因分析

doi:10.11686/cyxb2020486

摘要/Abstract

摘要：

分蘖与株高是禾本科草类植物重要的农艺性状，明确参与调控分蘖与株高的基因类型对牧草和草坪草分子辅助育种具有重要意义。以表型差异明显的两个高羊茅品种“Kentucky-31”（K31）和“Regenerate”为材料，旨在构建高羊茅分蘖节转录组图谱，挖掘在分蘖节部位调控生长发育相关的差异表达基因（differentially expressed genes，DEGs）。基于高通量测序技术平台Illumina HiSeq 2500×Miseq 300进行转录组测序，并将得到的数据进行de novo组装，结果共获得77872条单基因簇（unigene）。将获得的unigenes与非冗余蛋白数据库（non-redundant protein database，NR）、蛋白质数据库（universal protein，Uniprot）、基因本体数据库（gene ontology，GO）、东京基因与基因组数据库（kyoto encyclopedia of genes and genomes，KEGG）以及直系同源蛋白簇（clusters of orthologous groups，COG）数据库进行比对，结果显示：分别有59927、40213、44447、15146和13767条unigenes成功获得注释。“Regenerate”与“K31”对比有1573个上调DEGs和1441个下调DEGs。GO富集分析发现，DEGs主要富集在细胞、细胞组分、大分子复合物组装等生物过程。DEGs中共注释到42个差异表达转录因子，主要包括TCP、WRKY和ARF等19种类型。还注释到与8类植物激素相关的DEGs，包括生长素、细胞分裂素、脱落酸、赤霉素、乙烯、油菜素甾醇、水杨酸和茉莉酸。利用实时荧光定量PCR对DEGs进行表达模式验证，发现其与RNA-Seq测序结果一致，证实了测序结果的准确性。研究结果丰富了高羊茅的转录组序列资源，初步获得控制株高及分蘖发育的候选因子，为进一步开展基因功能及分子育种研究提供了理论支持。

关键词: 高羊茅, 转录组测序, 差异表达基因, 转录因子, 植物激素

Abstract:

Tiller number and plant height are important agronomic traits for grasses. Identification of genes controlling tiller development and plant height is of great significance for molecular assisted breeding of forage and turfgrass. In this study， two varieties of tall fescue （Festuca arundinacea） 'Kentucky-31' （K31） and 'Regenerate' were used to construct transcriptome maps and explore the differentially expressed genes （DEGs） regulating shoot growth and development. Transcriptomic sequencing was performed based on Illumina HiSeq 2500×Miseq 300， the data obtained were assembled de novo and a total of 77872 unigenes were obtained. The obtained unigenes were then blasted against the non-redundant protein （NR）， universal protein （Uniprot）， gene ontology （GO）， kyoto encyclopedia of genes and genomes （KEGG） and cluster of orthologous groups （COG） databases， and 59927， 40213， 44447， 15146 and 13767 unigenes， respectively， were successfully annotated. A total of 1573 up-regulated DEGs and 1441 down-regulated DEGs were detected in 'Regenerate' when compared to 'K31'. GO enrichment analysis found that DEGs are mainly enriched in biological processes such as cell and cell component and macromolecular complex assembly. A total of 42 differentially expressed transcription factors were noted among the identified DEGs， mainly belonging to teosinte branched 1， cycloidea， proliferating cell factors （TCP）， WRKY and auxin response factor （ARF） families. DEGs relating to eight plant hormone categories were also annotated， including auxin， cytokinin， abscisic acid， gibberellin， ethylene， brassinosteroids， salicylic acid and jasmonic acid. The expression pattern of DEGs verified by real-time PCR was consistent with RNA-Seq， indicating the RNA sequencing results were accurate. This study has advanced the transcriptome knowledge for tall fescue， obtained a preliminary identification of the candidate genes controlling plant height and tiller development， and provided theoretical support for further research on gene function and molecular breeding.

Key words: tall fescue, RNA-seq, differentially expressed genes, transcription factors, plant hormones

杨志民, 邢瑞, 丁鋆嘉, 庄黎丽. 基于转录组测序的高羊茅分蘖与株高相关差异表达基因分析[J]. 草业学报, 2022, 31(1): 145-163.

Zhi-min YANG, Rui XING, Yun-jia DING, Li-li ZHUANG. Analysis of differentially expressed genes in relation to tiller development and plant height based on transcriptomic sequencing of two tall fescue cultivars[J]. Acta Prataculturae Sinica, 2022, 31(1): 145-163.

图/表 14

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基因编号Unigene ID	正向引物（5′-3′）Forward primers （5′-3′）	反向引物（5′-3′）Reverse primers （5′-3′）
Unigene003394	GGTGGACGAGAAGCAGTACT	CGGGTAGTAGTTGCAGGTGA
Unigene054336	CTCAAGGGCAAGGAATCTGC	ATTCGATCGGGGAGAAAGCA
Unigene005703/4/5	AGAGTGTGATGGCGTTGAGA	GCAGATTCCTTGGCCTTGAG
Unigene005713	TGTCTCAACACCGTCACACT	ATGTTGAGGGAGATGGTGGC
Unigene074113	CAGAACTGGCAGAGCAACTC	GGGAGGAGGAAGACGACTAC
Unigene031989	CATCGTGCCAGTCAACTACAG	AGGTTGAAGTAGGAGTGGCC
Unigene055976	TCCCTCGTCAACTACCCCTA	TTGGGGAGCAGATGACACAT
Unigene063823	GCAAGCACCGATCCAGTATG	CGGACTTGTAGGATTTGGCG
Unigene036563	CTCAACTCCAACCGCTTCTG	GTTGTTGCTGAGGTCGAGC
Unigene067511	CTGCTCCTGTCCACCTGTAA	AGTCAGCGAAGAAGTCACCA
Unigene056675	GCGTCGTCACTGCTTTCTAT	CGTCGATGTACAGGTTGGTC
Unigene031117	CTCTCACTTGCCGTCTTTGG	GACGAGGCAGTGTCCATCT
Unigene059338	GAGCTGCCTTTCATCGCTAC	CTCTGGGATGGCAGAAAGGA
Unigene033181	GCTTACTCTCAGCCTCCACT	GGCCTGCAGAAATTGGGAAT
Unigene023003	TTGCTTTAGAAGGCAGGCAC	AGCAGCAAACACACCACTTT
Unigene033817	TGAAGCAGATGTGGAGCAGA	GCCTCCGCTTTATTGCTTGA
Unigene061235	AAGAACTGCATGCCGTTGTT	ATTCTTGGAGGCGTTCATGC
Unigene074427	CAACAAGGGTGCTGAGATCG	GGCGATGCTACCTTGTTCAG
Unigene037483	CTCGATAAGCTGTGGGACGA	CTAGTGCTGTGTTGATGGCC
Unigene003016	ATGGCATGTTCAACGGTGTC	GAGTATCTGTCCGTGGCTGA
Unigene066660	TCATCCCAGGTGAACACTCC	GCGCTGTACAAGTTCGGTAG
Unigene074432	ATCCGTCTCAGGTCCAAGTG	GCGAAATACTGTCCGCCAAT
Unigene022674	GACGGACGGAATCAAGAGGA	CATACCATTCGAAGTGCCGG
Unigene054725	GTAGCACCTCCGTCCCTATC	GGACTTGATCGAGCTGAGGA

基因编号Unigene ID	正向引物（5′-3′）Forward primers （5′-3′）	反向引物（5′-3′）Reverse primers （5′-3′）
Unigene003394	GGTGGACGAGAAGCAGTACT	CGGGTAGTAGTTGCAGGTGA
Unigene054336	CTCAAGGGCAAGGAATCTGC	ATTCGATCGGGGAGAAAGCA
Unigene005703/4/5	AGAGTGTGATGGCGTTGAGA	GCAGATTCCTTGGCCTTGAG
Unigene005713	TGTCTCAACACCGTCACACT	ATGTTGAGGGAGATGGTGGC
Unigene074113	CAGAACTGGCAGAGCAACTC	GGGAGGAGGAAGACGACTAC
Unigene031989	CATCGTGCCAGTCAACTACAG	AGGTTGAAGTAGGAGTGGCC
Unigene055976	TCCCTCGTCAACTACCCCTA	TTGGGGAGCAGATGACACAT
Unigene063823	GCAAGCACCGATCCAGTATG	CGGACTTGTAGGATTTGGCG
Unigene036563	CTCAACTCCAACCGCTTCTG	GTTGTTGCTGAGGTCGAGC
Unigene067511	CTGCTCCTGTCCACCTGTAA	AGTCAGCGAAGAAGTCACCA
Unigene056675	GCGTCGTCACTGCTTTCTAT	CGTCGATGTACAGGTTGGTC
Unigene031117	CTCTCACTTGCCGTCTTTGG	GACGAGGCAGTGTCCATCT
Unigene059338	GAGCTGCCTTTCATCGCTAC	CTCTGGGATGGCAGAAAGGA
Unigene033181	GCTTACTCTCAGCCTCCACT	GGCCTGCAGAAATTGGGAAT
Unigene023003	TTGCTTTAGAAGGCAGGCAC	AGCAGCAAACACACCACTTT
Unigene033817	TGAAGCAGATGTGGAGCAGA	GCCTCCGCTTTATTGCTTGA
Unigene061235	AAGAACTGCATGCCGTTGTT	ATTCTTGGAGGCGTTCATGC
Unigene074427	CAACAAGGGTGCTGAGATCG	GGCGATGCTACCTTGTTCAG
Unigene037483	CTCGATAAGCTGTGGGACGA	CTAGTGCTGTGTTGATGGCC
Unigene003016	ATGGCATGTTCAACGGTGTC	GAGTATCTGTCCGTGGCTGA
Unigene066660	TCATCCCAGGTGAACACTCC	GCGCTGTACAAGTTCGGTAG
Unigene074432	ATCCGTCTCAGGTCCAAGTG	GCGAAATACTGTCCGCCAAT
Unigene022674	GACGGACGGAATCAAGAGGA	CATACCATTCGAAGTGCCGG
Unigene054725	GTAGCACCTCCGTCCCTATC	GGACTTGATCGAGCTGAGGA

样品Sample	原始数据Raw reads	测序数据Clean reads	原始下机数据Raw bases	测序下机数据Clean bases	测序数据比率 Clean data rate （%）
Regenerate	88475512	88345910	11834878498	11576251799	97.81
K31	76673214	76533900	10284187805	10037407880	97.60

样品Sample	原始数据Raw reads	测序数据Clean reads	原始下机数据Raw bases	测序下机数据Clean bases	测序数据比率 Clean data rate （%）
Regenerate	88475512	88345910	11834878498	11576251799	97.81
K31	76673214	76533900	10284187805	10037407880	97.60

RNA-Seq信息 RNA-Seq information	数据 Data
Unigene 数目 Unigene number	77872
最大unigene bases Max unigene bases （bp）	13402
最小unigene bases Min unigene bases （bp）	200
全部数据长度 Whole dataset lengths （bp）	47586469
平均unigene长度 Average unigene lengths （bp）	611.086
（G+C）/（A+T+G+C）（%）	53.2
N₅₀ （bp）	920
N₉₀ （bp）	262