两种砧木楸树嫁接苗生长差异及转录组比较分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1557

摘要/Abstract

摘要：

嫁接是楸树（Catalpa bungei）良种扩繁的主要途径。为探讨以梓树和滇楸为砧木的两种‘苏楸1号’良种当年生嫁接苗生长差异，比较了二者的嫁接成活率、新梢长度、新梢粗度、接穗粗度/砧木粗度、叶面积等，以筛选较佳砧木，并通过高通量测序平台对两种嫁接苗叶片和顶芽进行转录组测序分析，采用实时荧光定量PCR技术对初步筛选的差异表达基因进行验证。结果显示，以梓树为砧木的嫁接成活率和嫁接苗各生长量均显著高于以滇楸为砧木的嫁接苗；转录组测序筛选出两个组合叶片和顶芽差异表达基因共计559个，其中上调表达基因192个，下调表达基因367个；GO富集分析表明559个差异表达基因显著富集在代谢过程、细胞过程、细胞器、结合和催化活性等功能类别；KEGG富集分析表明叶片中213个差异表达基因显著富集到66条代谢途径，顶芽中137个差异表达基因显著富集到45条代谢途径；初步筛选出的Unigene0040270、Unigene0006320、Unigene0036149和Unigene0007805等4个差异表达基因的RT-qPCR趋势与转录组数据一致。本研究结果发现两种嫁接苗的生长存在显著差异，其中以梓树为砧木的嫁接苗各生长参数更好，同时在转录组角度上对其差异进行了初步解析，为楸树嫁接分子研究提供一定的参考价值。

关键词: 嫁接, 楸树, 生长差异, 转录组, 差异表达基因

Abstract:

Grafting is the main way of reproducing and expanding Catalpa bunge of superior cultivar. In order to investigate the growth differences of two ‘Suqiu No. 1’ superior cultivars, taking Catalpa ovata and Catalpa fargesii as rootstocks, the survival rate, new shoot length, new shoot thickness, scion diameter/stock diameter, leaf area of the two cultivars were studied for screening the better rootstock. Transcriptome sequencing was performed on the leaves and terminal buds of the two grafted seedlings by high-throughput sequencing platform. The differentially expressed genes were confirmed by quantitative real-time PCR. The results showed that the survival rate and the growth of the grafted seedlings with C. ovata as the rootstock were significantly higher than those with C. fargesii as the rootstock. Total 559 differentially expressed genes were screened by transcriptome sequencing, including total 192 up-regulated genes and total 367 down-regulated genes. GO enrichment analysis showed that total 559 differentially expressed genes were significantly enriched in metabolic process, cellular process, organelles, binding and catalytic activities; KEGG enrichment analysis showed that total 213 differentially expressed genes were significantly enriched to total 66 metabolic pathways in leaves, and total 137 differentially expressed genes were significantly enriched to total 45 metabolic pathways in terminal buds. The RT-qPCR trends of four differentially expressed genes（Unigene0040270, Unigene0006320, Unigene0036149 and Unigene0007805）were consistent with the transcriptome data. The results of this study showed that there were significant differences in the growth of the two grafted seedlings, among which the growth parameters of the grafted seedlings with C. fargesii as the rootstock were better. At the same time, the differences were preliminarily analyzed from the perspective of transcriptome, providing a certain reference value for the study of C. bungei grafting molecules.

Key words: grafting, Catalpa bungei, growth differences, transcriptome, differentially expressed genes

付钰, 贾瑞瑞, 何荷, 王良桂, 杨秀莲. 两种砧木楸树嫁接苗生长差异及转录组比较分析[J]. 生物技术通报, 2023, 39(8): 251-261.

FU Yu, JIA Rui-rui, HE He, WANG Liang-gui, YANG Xiu-lian. Growth Differences Among Grafted Seedlings with Two Rootstocks of Catalpa bungei and Comparative Analysis of Transcriptome[J]. Biotechnology Bulletin, 2023, 39(8): 251-261.

图/表 13

表1 嫁接组合及取样部位

Table1 Grafting combination and sampling part

嫁接组合 Grafting combination	取样部位 Sampling part	编号 Numbering
苏楸1号/梓树 ‘Suqiu No. 1’/ Catalpa ovata	叶片Leaf	ZS-L
苏楸1号/梓树 ‘Suqiu No. 1’/ Catalpa ovata	顶芽Bud	ZS-B
苏楸1号/滇楸 ‘Suqiu No. 1’/Catalpa fargesii	叶片Leaf	DS-L
苏楸1号/滇楸 ‘Suqiu No. 1’/Catalpa fargesii	顶芽Bud	DS-B

表2 RT-qPCR引物序列

Table 2 Primer sequences of RT-qPCR

引物名称Primer name	序列Sequence（5'-3'）
CfMADH-F	AGCTTCCATTCTTTGCCTCA
CfMADH-R	TCCGAACAAAAGCAATACCC
Unigene0036149-F	AGGAACAGGAGAATGTGAAGAGG
Unigene0036149-R	CCGTCGTCACTTCTCGCAG
Unigene0007805-F	CAAAAAATGCTGGAAAAGTACCC
Unigene0007805-R	TGGTAAAATCGTGGTTCTCAAGTG
Unigene0040270-F	CCGCTTCATCTTGGTTCAGTG
Unigene0040270-R	AAGAAGGAAGATTTGCTGTTGGA
Unigene0006320-F	AGAAACACGAAAGTGAATGCTAAAC
Unigene0006320-R	GCACTCGGATGAAACGAAAAC

表3 两个楸树嫁接苗的成活率和生长量

Table 3 Survival rates and growths of grafted seedlings of two Catalpa trees

嫁接组合 Grafting combination	成活率 Survival rate/%	新梢长度 New shoot length/cm	新梢粗度 New shoot thickness/cm	接穗粗度/砧木粗度 Scion diameter/Stock diameter	叶面积 Leaf area/cm²
ZS	60.95±0.064**	107.51±12.25**	2.47±0.65**	0.822±0.029**	338.64±6.11**
DS	32.05±0.038	83.43±5.06	2.04±1.57	0.721±0.087	247.61±6.59

表4 12个样本测序数据的汇总结果

Table 4 Summary results of sequencing data for 12 samples

高质量数据碱基总数 Clean data/bp	Q20/%	Q30/%	N/%	GC/%
75 612 886 298	96.65-97.36	91.16-92.69	0	43.79-44.57

图1 样本间基因表达水平相关性热图

Fig. 1 Correlation heat map of gene expression levels among samples

图2 不同嫁接组合差异基因火山图横坐标表示两个分组间的差异倍数log2值，纵坐标表示两个分组差异的FDR的负Log10值，红色代表表达量上调，蓝色代表表达量下调

Fig. 2 Volcano plot of different genes in different grafting combinations The abscissa indicates the multiple log2 value of the difference between the two groups, the ordinate indicates the negative Log10 value of the FDR of the difference between the two groups. Red indicates the expression was up-regulated and blue was down-regulated

图3 不同嫁接组合差异基因的GO注释富集分析图 A：ZS-L vs DS-L；B：ZS-B vs DS-B。横坐标表示GO Term level 1与2，纵坐标表示差异表达基因的数量

Fig. 3 GO annotation enrichment analysis diagram of differential genes in different grafting combinations A：ZS-LvsDS-L; B：ZS-BvsDS-B. The abscissa represents GO Term level 1 and 2, the ordinate represents the number of differentially expressed genes

图4 不同嫁接组合显著差异基因KEGG富集分析图 A：ZS-L vs DS-L；B：ZS-B vs DS-B；第一圈：富集前20的pathway，圈外为差异基因数目的坐标尺。不同的颜色代表不同的A class；第二圈：差异基因背景中该pathway的数目以及Q值。差异基因背景数量越多条形越长，Q值越小颜色越红；第三圈：上下调差异基因比例条形图，深紫色代表上调差异基因比例，浅紫色代表下调差异基因比例；下方显示具体的数值；第四圈：各pathway的RichFactor值（该pathway中差异基因数量除以该pathway中所有数量），背景网格线，每一格代表0.1

Fig. 4 KEGG annotation enrichment analysis diagram of differential genes in different grafting combinations A：ZS-L vs DS-L；B：ZS-B vs DS-B. The first circle: enrich the top 20 pathways, outside the circle is the coordinate ruler of the number of differential genes. Different colors indicate different A classes. The second circle: the number and Q value of the pathway in the differential gene background. The more the number of differential genes background, the longer the bar, the smaller the Q value, the redder the color. The third circle: the bar graph of the ratio of up- and down-regulated differential genes, dark purple represents the ratio of up-regulated differential genes, and light purple represents the ratio of down-regulated differential genes; the lower part shows the specific value. The fourth circle: the RichFactor value of each pathway（the number of differential genes in the pathway divided by all the numbers in the pathway）, background grid lines, each grid indicates 0.1

图5 光合相关代谢通路

Fig. 5 Photosynthesis-related metabolic pathway

图6 淀粉和蔗糖代谢相关通路

Fig. 6 Pathways related to starch and sucrose metabolism

表5 与光合作用相关的差异表达基因统计

Tab.5 Statistics of differentially expressed genes related to photosynthesis

通路Pathway	基因ID Unigene ID	基因名称Symbol	描述[ID]Description[ID]
类胡萝卜素生物合成 Carotenoid biosynthesis	Unigene0051519	PSY1	顺式八氢番茄红素合酶[EC:2.5.1.32]15-cis-Phytoene synthase
	Unigene0046587	LCY1	番茄红素β-环化酶[EC:5.5.1.19]Lycopene beta-cyclase
	Unigene0013377	NCED2	9-顺式环氧类胡萝卜素双加氧酶[EC:1.13.11.51] 9-cis-Epoxycarotenoid dioxygenase
	Unigene0044139	CYP707A2	脱落酸-8'-羟化酶[EC:1.14.14.137]Abscisic acid 8'-hydroxylase
卟啉和叶绿素代谢 Porphyrin and chlorophyll metabolism	Unigene0054531	HEMA1	谷氨酰-tRNA还原酶[EC:1.2.1.70]Glutamyl-tRNA reductase
	Unigene0017692	CRD1	镁-原卟啉IX单甲酯（氧化）环化[EC:1.14.13.81] Magnesium-protoporphyrin IX monomethyl ester（oxidative）cyclase
	Unigene0005359	CAO	叶绿素a加氧酶[EC:1.14.13.122]Chlorophyllide a oxygenase
	Unigene0040270	CAO	叶绿素a加氧酶[EC:1.14.13.122]Chlorophyllide a oxygenase

表6 与淀粉和蔗糖代谢相关的差异表达基因统计

Tab.6 Statistics of differentially expressed genes related to starch and sucrose metabolism

通路Pathway	基因ID Unigene ID	基因名称Symbol	描述[ID]Description[ID]
淀粉和蔗糖代谢 Starch and sucrose metabolism	Unigene0038167	SPS2	蔗糖-磷酸合酶[EC:2.4.1.14]Sucrose-phosphate synthase
	Unigene0005917	SS2	淀粉合酶[EC:2.4.1.21]Starch synthase
	Unigene0017971	TPPJ	海藻糖6-磷酸磷酸酶[EC:3.1.3.12]Trehalose 6-phosphate phosphatase
	Unigene0000390	GLU3	内切葡聚糖酶[EC:3.2.1.4]Endoglucanase
	Unigene0022047	GLU1	β-葡萄糖苷酶[EC:3.2.1.21]beta-Glucosidase
	Unigene0048299	INVDC4*	β-果糖呋喃糖苷酶[EC:3.2.1.26]beta-Fructofuranosidase
	Unigene0006320	WAXY	颗粒结合淀粉合酶[EC:2.4.1.242]Granule-bound starch synthase
	Unigene0048994	STP-1	糖原磷酸化酶[EC:2.4.1.1]Glycogen phosphorylase
	Unigene0047773	BAM1	β-淀粉酶[EC:3.2.1.2]beta-Amylase
	Unigene0043824	BACOVA-02659	β-葡萄糖苷酶[EC:3.2.1.21]beta-Glucosidase

图7 四个基因在两个嫁接组合中的相对表达量不同字母表示不同嫁接组合相对表达量差异显著（P<0.05）

Fig. 7 Relative expressions of four genes in two grafting combinations Different letters indicate the significant difference of relative expression at different grafting combinations.（P<0. 05）

参考文献 37

[1]	Liu YM, Zhou L, Zhu YQ, et al. Anatomical features and its radial variations among different Catalpa bungei clones[J]. Forests, 2020, 11(8): 824. doi: 10.3390/f11080824 URL
[2]	Lu N, Zhang MM, Xiao Y, et al. Construction of a high-density genetic map and QTL mapping of leaf traits and plant growth in an interspecific F1 population of Catalpa bungei × Catalpa duclouxii Dode[J]. BMC Plant Biol, 2019, 19(1): 596. doi: 10.1186/s12870-019-2207-y
[3]	Wu JW, Li JY, Su Y, et al. A morphophysiological analysis of the effects of drought and shade on Catalpa bungei plantlets[J]. Acta Physiol Plant, 2017, 39(3): 80. doi: 10.1007/s11738-017-2380-2 URL
[4]	Xiao Y, Wang JH, Yun HL, et al. Genetic evaluation and combined selection for the simultaneous improvement of growth and wood properties in Catalpa bungei clones[J]. Forests, 2021, 12(7): 868. doi: 10.3390/f12070868 URL
[5]	王改萍, 王良桂, 王晓聪, 等. 楸树嫩枝扦插生根发育及根系特征分析[J]. 南京林业大学学报: 自然科学版, 2020, 44(6): 94-102.
	Wang GP, Wang LG, Wang XC, et al. Dynamic characteristics of cutting rooting of Catalpa bungei with tender branches[J]. J Nanjing For Univ Nat Sci Ed, 2020, 44(6): 94-102.
[6]	Chen W, Meng PP, Feng H, et al. Effects of arbuscular mycorrhizal fungi on growth and physiological performance of Catalpa bungei C.A.Mey. under drought stress[J]. Forests, 2020, 11(10): 1117. doi: 10.3390/f11101117 URL
[7]	Lin J, Wu LH, Jing L, et al. Effect of different plant growth regulators on callus induction in Catalpa bungei[J]. Afr J Agric Res, 2010, 5: 2699-2704.
[8]	Miao L, Li Q, Sun TS, et al. Sugars promote graft union development in the heterograft of cucumber onto pumpkin[J]. Hortic Res, 2021, 8(1): 146. doi: 10.1038/s41438-021-00580-5
[9]	Ren Y, Xu Q, Wang LW, et al. Involvement of metabolic, physiological and hormonal responses in the graft-compatible process of cucumber/pumpkin combinations was revealed through the integrative analysis of mRNA and miRNA expression[J]. Plant Physiol Biochem, 2018, 129: 368-380. doi: 10.1016/j.plaphy.2018.06.021 URL
[10]	Karaca F, Yetİşİr H, Solmaz İ, et al. Rootstock potential of Turkish Lagenaria siceraria germplasm for watermelon: plant growth, yield and quality[J]. Turkish J Agric For, 2012: 167-177.
[11]	Rasool A, Mansoor S, Bhat KM, et al. Mechanisms underlying graft union formation and rootstock scion interaction in horticultural plants[J]. Front Plant Sci, 2020, 11: 590847. doi: 10.3389/fpls.2020.590847 URL
[12]	Świerczyński S. The effect of rootstock activity for growth and root system soaking in Trichoderma atroviride on the graft success and continued growth of beech(Fagus sylvatica L.) plants[J]. Agronomy, 2022, 12(6): 1259. doi: 10.3390/agronomy12061259 URL
[13]	Liu WQ, Xiang CG, Li XJ, et al. Identification of long-distance transmissible mRNA between scion and rootstock in cucurbit seedling heterografts[J]. Int J Mol Sci, 2020, 21(15): 5253. doi: 10.3390/ijms21155253 URL
[14]	Kaseb MO, Umer MJ, Anees M, et al. Transcriptome profiling to dissect the role of genome duplication on graft compatibility mechanisms in watermelon[J]. Biology, 2022, 11(4): 575. doi: 10.3390/biology11040575 URL
[15]	Liu XY, Li J, Liu MM, et al. Transcriptome profiling to understand the effect of Citrus rootstocks on the growth of ‘shatangju’ mandarin[J]. PLoS One, 2017, 12(1): e0169897. doi: 10.1371/journal.pone.0169897 URL
[16]	Davoudi M, Song MF, Zhang MR, et al. Long-distance control of the scion by the rootstock under drought stress as revealed by transcriptome sequencing and mobile mRNA identification[J]. Hortic Res, 2022, 9: uhab033. doi: 10.1093/hr/uhab033 URL
[17]	申妍颖, 李雪涵, 李飞鸿, 等. 中间砧‘南通小方柿’嫁接柿转录组测序及分析[J]. 分子植物育种, 2019, 17(5): 1454-1466.
	Shen YY, Li XH, Li FH, et al. Transcriptome sequencing and analysis of grafted persimmon with ‘Nantong-Xiaofangshi’ as the interstock[J]. Mol Plant Breed, 2019, 17(5): 1454-1466.
[18]	Li GF, Ma JJ, Tan M, et al. Transcriptome analysis reveals the effects of sugar metabolism and auxin and cytokinin signaling pathways on root growth and development of grafted apple[J]. BMC Genom, 2016, 17(1): 150. doi: 10.1186/s12864-016-2484-x URL
[19]	Young MD, Wakefield MJ, Smyth GK, et al. Gene ontology analysis for RNA-seq: accounting for selection bias[J]. Genome Biol, 2010, 11(2): R14. doi: 10.1186/gb-2010-11-2-r14 URL
[20]	Mao XZ, Cai T, Olyarchuk JG, et al. Automated genome annotation and pathway identification using the KEGG Orthology(KO)as a controlled vocabulary[J]. Bioinformatics, 2005, 21(19): 3787-3793. doi: 10.1093/bioinformatics/bti430 URL
[21]	杨英英, 赵林姣, 杨桂娟, 等. ‘麦缘锦楸’叶色表型RT-qPCR内参基因筛选及验证[J]. 林业科学研究, 2022, 35(1): 123-131.
	Yang YY, Zhao LJ, Yang GJ, et al. Selection and validation of reference genes for leaf color phenotype in ‘maiyuanjinqiu’, a Catalpa fargesii variety, by RT-qPCR[J]. For Res, 2022, 35(1): 123-131.
[22]	He W, Xie R, Wang Y, et al. Comparative transcriptomic analysis on compatible/incompatible grafts in citrus[J]. Hortic Res, 2022, 9: uhab072. doi: 10.1093/hr/uhab072 URL
[23]	钟亚琴. 嫁接西瓜响应低钾胁迫的生理和分子机制研究[D]. 武汉: 华中农业大学, 2019.
	Zhong YQ. Physiological and molecular mechanism of grafted watermelon in response to low potassium stress[D]. Wuhan: Huazhong Agricultural University, 2019.
[24]	Thomas A, Brauer D, Sauer T, et al. Cultivar influences early rootstock and scion survival of grafted black walnut[J]. J Am Pomol Soc, 2008, 62(1): 3-12.
[25]	Reig G, Zarrouk O, Font i Forcada C, et al. Anatomical graft compatibility study between apricot cultivars and different plum based rootstocks[J]. Sci Hortic, 2018, 237: 67-73. doi: 10.1016/j.scienta.2018.03.035 URL
[26]	Huang Y, Zhao LQ, Kong QS, et al. Comprehensive mineral nutrition analysis of watermelon grafted onto two different rootstocks[J]. Hortic Plant J, 2016, 2(2): 105-113.
[27]	Bhuiyan MFA, Rahim MA, Alam MS. Study on the growth of plants produced by epicotyl(stone)grafting with different rootstock scion combinations in mango[J]. J Agrofor Environ, 2010, 3(2): 163-166.
[28]	Harris ZN, Awale M, Bhakta N, et al. Multi-dimensional leaf phenotypes reflect root system genotype in grafted grapevine over the growing season[J]. GigaScience, 2021, 10(12): giab087. doi: 10.1093/gigascience/giab087 URL
[29]	Yang YJ, Lu XM, Yan B, et al. Bottle gourd rootstock-grafting affects nitrogen metabolism in NaCl-stressed watermelon leaves and enhances short-term salt tolerance[J]. J Plant Physiol, 2013, 170(7): 653-661.
[30]	Somkuwar RG, Bahetwar A, Khan I, et al. Changes in growth, photosynthetic activities, biochemical parameters and amino acid profile of Thompson Seedless grapes(Vitis vinifera L.)[J]. J Environ Biol, 2014, 35(6): 1157-1163. pmid: 25522520
[31]	Proebsting WM, Maggard SP, Guo WW. The relationship of thiamine to the Alt locus of Pisum sativum L[J]. J Plant Physiol, 1990, 136(2): 231-235. doi: 10.1016/S0176-1617(11)81671-8 URL
[32]	Sayed SA, Gadallah MAA. Effects of shoot and root application of thiamin on salt-stressed sunflower plants[J]. Plant Growth Regul, 2002, 36(1): 71-80. doi: 10.1023/A:1014784831387 URL
[33]	Xu Q, Guo SR, Li L, et al. Proteomics analysis of compatibility and incompatibility in grafted cucumber seedlings[J]. Plant Physiol Biochem, 2016, 105: 21-28. doi: 10.1016/j.plaphy.2016.04.001 URL
[34]	Kim SH, Kim YH, Ahn YO, et al. Downregulation of the lycopene ϵ-cyclase gene increases carotenoid synthesis via the β-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli[J]. Physiol Plant, 2013, 147(4): 432-442. doi: 10.1111/ppl.2013.147.issue-4 URL
[35]	Komatsu A, Takanokura Y, Moriguchi T, et al. Differential expression of three sucrose-phosphate synthase isoforms during sucrose accumulation in citrus fruits(Citrus unshiu Marc.)[J]. Plant Science, 1999, 140(2): 169-178. doi: 10.1016/S0168-9452(98)00217-9 URL
[36]	Zhu YJ, Moore PH. Sucrose accumulation in the sugarcane stem is regulated by the difference between the activities of soluble acid invertase and sucrose phosphate synthase[J]. Plant Physiol, 1997, 115(2): 609-616. doi: 10.1104/pp.115.2.609 pmid: 12223829
[37]	Pauly M, Keegstra K. Plant cell wall polymers as precursors for biofuels[J]. Curr Opin Plant Biol, 2010, 13(3): 304-311. doi: 10.1016/j.pbi.2009.12.009 URL