中国农业科学 ›› 2022, Vol. 55 ›› Issue (3): 438-450.doi: 10.3864/j.issn.0578-1752.2022.03.002
石习1,2(),宁丽华2,葛敏2,邬奇2,赵涵1,2,*(
)
收稿日期:
2021-07-30
接受日期:
2021-10-18
出版日期:
2022-02-01
发布日期:
2022-02-11
通讯作者:
赵涵
作者简介:
石习,Tel:13813928391;E-mail: 基金资助:
SHI Xi1,2(),NING LiHua2,GE Min2,WU Qi2,ZHAO Han1,2,*(
)
Received:
2021-07-30
Accepted:
2021-10-18
Online:
2022-02-01
Published:
2022-02-11
Contact:
Han ZHAO
摘要: 【背景】 RNA表达丰度作为一种生物标记物已广泛应用于临床诊断阶段,但在农业栽培中诊断作物营养状况的应用较少。 【目的】 挖掘和验证转录水平上可以作为生物标记物精确指示玉米氮营养状况的基因,指导精准施用氮肥。【方法】 基于不同氮素处理的基因芯片和RNA-Seq数据,通过生物信息学和统计学方法初步筛选出基因表达丰度高度响应氮素处理的生物标记物候选基因;利用不同基因型、不同氮处理的玉米材料,通过荧光定量PCR方法和凯氏定氮法,进一步筛选氮响应生物标记物基因;并构建预测玉米氮状况的广义线性模型,准确指示玉米氮营养状况。 【结果】 首先初步筛选出10个表达水平较高的基因,且mRNA表达丰度高度响应氮素处理的生物标记物候选基因;利用不同氮素条件下种植的B73材料从10个候选基因中进一步筛选出8个在充足氮、限制氮处理后基因表达丰度存在显著差异的基因;随后选取遗传多样性丰富、生态区域广泛的26种自交系材料和4种杂交种材料进一步筛选,发现有4个基因表达独立于基因型,可以在不同基因型玉米材料中稳定表达;并且通过相关性分析发现,在充足氮和限制氮处理下,这4个基因表达丰度差异与穗位叶总氮含量差异具有显著相关性(R2均大于0.6),表明这4个基因可以作为氮响应生物标记物进行实际应用;将4个氮响应生物标记物基因分别组合构建两基因、三基因、四基因线性模型,由Zm00001d024281(X2)、Zm00001d039049(X3)和Zm00001d037680(X4)这三个基因构建的线性模型用于预测玉米植株氮状况的应用性最强,其函数关系为Y=1.143+0.017X2–0.302X3+0.017X4;选取大田种植的6个杂交种材料对三基因模型预测功能进行验证,结果表明,三基因模型能够在大田环境中准确诊断玉米植株氮素营养状况。【结论】 获得4个高度响应玉米氮状况的生物标记物基因,构建的三基因模型可以准确预测玉米氮素营养状况。该生物标记物的开发可以有效实时监测玉米植株氮状态,优化氮肥使用,从而实现成本最低时农作物产量的最大化。
石习, 宁丽华, 葛敏, 邬奇, 赵涵. 玉米氮状况相关生物标记物的筛选和应用[J]. 中国农业科学, 2022, 55(3): 438-450.
SHI Xi, NING LiHua, GE Min, WU Qi, ZHAO Han. Screening and Application of Biomarkers Related to Maize Nitrogen Status[J]. Scientia Agricultura Sinica, 2022, 55(3): 438-450.
表1
本研究所用的引物序列"
基因编号 Gene No. | 基因 ID Gene ID | 正向引物序列 Forward primer sequence (5′-3′) | 反向引物序列 Reverse primer sequence (5′-3′) |
---|---|---|---|
NM01 | Zm00001d009599 | TCAAGTCTGCCGAGGAGGTG | CGGGTCCAGAATCCCTGTGTC |
NM02 | Zm00001d022630 | ACACCGTACCGTCGTCTCTG | TCGTTGGGCATCATATCACAGTG |
NM03 | Zm00001d024281 | TGGTCCGACAGGTTCTACAAGG | AGTGCTCGCTGGTGTGCTC |
NM04 | Zm00001d045519 | GAAGAAGGGCACCACCAT | TTCTCTGGATCGTCTCCCT |
NM05 | Zm00001d026696 | TGGATGAAGCAGCTGATGTT | GAAATTCTTGAAGGCGTGGATG |
NM06 | Zm00001d039049 | CTACGGCGAGGGCACATCC | GACAGACAGACAGAGGTCCATCC |
NM07 | Zm00001d037680 | CGCACCGAGGACCAGAGG | CAGCGCACCTCCTCAGCAG |
NM08 | Zm00001d042867 | GCGACCAGTGCGGCATTTG | ATCATCAGTACGACGGGTGCAG |
NM09 | Zm00001d003924 | GTCGCCTACGTCGAGTTC | GTTGCGTGTAGATGAAGTTGTC |
NM10 | Zm00001d002052 | ATTGCCATCCACGGCGG | TGCGCGAAGCGGAGGAGGA |
NM11 | Zm00001d006438 | CACCCGGTTGGCTATGCTGTAC | TGTGCTCCACCAGAAGGCTGAC |
表2
氮响应生物标记物候选基因的初步筛选"
阶段 Phase | 数据处理步骤 Data-processing step | 输入探针数量 No. of input probe set | 输出探针数量 No. of output probe set |
---|---|---|---|
数据过滤 Data filtering | (1)Log2强度值>9.0 Log2 intensity>9.0 | 84246 | 11891 |
(2)t-test P<0.01 | 11891 | 6632 | |
(3)基因表达强度差异>10或<0.1 The fold difference of gene expression>10 or <0.1 | 6632 | 69 | |
氮响应生物标记物候选基因筛选 Screened the candidate genes for nitrogen responsive biomarkers | (1)筛选在2组数据中均显著响应氮处理的候选基因 Screened the candidate genes that significantly responded to nitrogen treatment in both groups of data | 63 | 25 |
(2)MAIZE GDB筛选叶片中表达的候选基因 Screened candidate genes expressed in leaves by MAIZE GDB | 25 | 10 |
表3
4个生物标记物候选基因"
基因编号 Gene No. | 基因ID Gene ID | 功能 Function | N0处理后基因表达丰度变化 Changes of gene expression abundance under N0 treatment |
---|---|---|---|
NM02 | Zm00001d022630 | 编码KNOTTED INDUCED1蛋白 Coding KNOTTED INDUCED 1 protein | 下调 Down-regulated |
NM03 | Zm00001d024281 | 编码多胺氧化酶1 Coding POLYAMINE OXIDASE 1 | 下调 Down-regulated |
NM06 | Zm00001d039049 | 编码MYB蛋白Coding MYB protein | 上调 Up-regulated |
NM07 | Zm00001d037680 | 编码营养贮藏蛋白2 Coding VEGETATIVE STORAGE PROTEIN 2 | 下调 Down-regulated |
表4
模型函数关系"
广义线性模型 Generalized linear models | 函数关系 Function relationship | 相关性 Correlation (R2) | P值 P value |
---|---|---|---|
两基因模型 The two-genes model | Y=1.152–0.306X3+0.020X4 | 0.51 | 7.283E-9 |
三基因模型 The three-genes model | Y=1.143+0.017X2–0.302X3+0.017X4 | 0.53 | 1.870E-8 |
四基因模型 The four-genes model | Y=1.142+0.020X2–0.303X3+0.017X4 | 0.53 | 8.411E-8 |
[1] | YAN X, JIN J Y, LIANG M Z. Grain crop fertilization status and factors influencing farmers’ decision making on fertilizer use: China case study. Agricultural Science & Technology, 2016, 17(10):2394-2440. |
[2] | 赵英杰. 花生—春玉米轮作的肥料效应及减量优化施肥技术研究[D]. 保定: 河北农业大学, 2019. |
ZHAO Y J. Study on fertilizer effect and reduction fertilization technology of peanut-spring maize rotation[D]. Baoding: Hebei Agricultural University, 2019. (in Chinese) | |
[3] |
GUO J H, LIU X J, ZHANG Y, SHEN J L, HAN W X, ZHANG W F, CHRISTIE P, GOULDING K W T, VITOUSEK P M, ZHANG F S. Significant acidification in major Chinese croplands. Science, 2010, 327(5968):1008-1010.
doi: 10.1126/science.1182570 |
[4] |
LIU X J, ZHANG Y, HAN W X, TANG A H, SHEN J L, CUI Z L, VITOUSEK P, ERISMAN J W, GOULDING K, CHRISTIE P, FANGMEIER A, ZHANG F S. Enhanced nitrogen deposition over China. Nature, 2013, 494(7438):459-462.
doi: 10.1038/nature11917 |
[5] |
ELLIOTT D E, REUTER D J, GROWDEN B, SCHULTZ J E, MUHLHAN P H, HEANES J G. Improved strategies for diagnosing and correcting nitrogen deficiency in spring wheat. Journal of Plant Nutrition, 1987, 10(9):1761-1770.
doi: 10.1080/01904168709363716 |
[6] | PATTON C J, TRUITT E P. Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; determination of the total phosphorus by a Kjeldahl digestion method and an automated colorimetric finish that includes dialysis. Journal of the American Medical Association, 1992, 15(39):92-146. |
[7] | 宋文冲, 胡春胜, 程一松, 代辉. 作物氮素营养诊断方法研究进展. 土壤通报, 2006(2):2369-2372. |
SONG W C, HU C S, CHENG Y S, DAI H. Research progress on diagnostic methods of crop nitrogen nutrition. Chinese Journal of Soil Science, 2006(2):2369-2372. (in Chinese) | |
[8] |
WOOD C W, TRACY P W, REEVES D W, EDMISTEN K L. Determination of cotton nitrogen status with a hand-held chlorophyll meter. Journal of Plant Nutrition, 1992, 15(9):1435-1448.
doi: 10.1080/01904169209364409 |
[9] |
TURNER F T, JUND M F. Chlorophyll meter to predict nitrogen top dress requirement for semidwarf rice. Agronomy Journal, 1991, 83(5):926-928.
doi: 10.2134/agronj1991.00021962008300050029x |
[10] | FOLLETT R H, FOLLETT R F, HALVORSON A D. Use of a chlorophyll meter to evaluate the nitrogen status of dryland winter wheat. Communications in Soil Science & Plant Analysis, 1992, 23(7/8):687-697. |
[11] |
CHAPMAN S C, BARRETO H J. Using a chlorophyll meter to estimate specific leaf nitrogen of tropical maize during vegetative growth. Agronomy Journal, 1997, 89(4):557-562.
doi: 10.2134/agronj1997.00021962008900040004x |
[12] | 唐延林, 王人潮, 张金恒, 王珂. 高光谱与叶绿素计快速测定大麦氮素营养状况研究. 麦类作物学报, 2003, 23(1):63-66. |
TANG Y L, WANG R C, ZHANG J H, WANG K. Study on determining nitrogenous levels of barley by hyperspectral and chlorophyll meter. Journal of Triticeae Crops, 2003, 23(1):63-66. (in Chinese) | |
[13] |
TURNER F T, JUND M F. Assessing the nitrogen requirements of rice crops with a chlorophyll meter method. Australian Journal of Experimental Agriculture, 1994, 34(7):1001-1005.
doi: 10.1071/EA9941001 |
[14] | OLAV H B, SOLHAUG K A. Effect of irradiance on chlorophyll estimation with the minolta SPAD-502 leaf chlorophyll meter. Annals of Botany, 1998(3):389-392. |
[15] |
SATHYABAARATHI R, USHASHI B, DEVI D G, ROOPARANI K, CHANDRANI T, DIPSHIKHA C, NARAYANASWAMY B K, AMIT S, NAGASUMA C. VB10, a new blood biomarker for differential diagnosis and recovery monitoring of acute viral and bacterial infections. EBioMedicine, 2021, 67(2):103352.
doi: 10.1016/j.ebiom.2021.103352 |
[16] | GWENAELLE M, MAYJONADE B, DIDIER V. A biomarker based on gene expression indicates plant water status in controlled and natural environments. Plant Cell & Environment, 2013, 36(12):2175-2189. |
[17] |
KENKEL C D, SHERIDAN C, LEAL M C, BHAGOOLI R, CASTILLO K D, KURATA N, MCGINTY E, GOULET T L, MATZ M V. Diagnostic gene expression biomarkers of coral thermal stress. Molecular Ecology Resources, 2014, 14(4):667-678.
doi: 10.1111/men.2014.14.issue-4 |
[18] |
YANG X F, WU J R, ZIEGLER T E, YANG X, ZAYED A, RAJANI M S, ZHOU D F, BASRA A S, SCHACHTMAN D P, PENG M S, ARMSTRONG C L, CALDO R A, MORRELL J A, LACY M, STAUB J M. Gene expression biomarkers provide sensitive indicators of in planta nitrogen status in maize. Plant Physiology, 2011, 157(4):1841-1852.
doi: 10.1104/pp.111.187898 |
[19] |
葛敏, 吕远大, 张体付, 周玲, 林峰, 赵涵. 玉米氮素敏感性差异自交系的表达谱分析. 作物学报, 2016, 42(10):1487-1494.
doi: 10.3724/SP.J.1006.2016.01487 |
GE M, LÜ Y D, ZHANG T F, ZHOU L, LIN F, ZHAO H. Global transcriptome analysis in high- and low-nitrogen responsive inbred lines of maize. Acta Agronomica Sinica, 2016, 42(10):1487-1494. (in Chinese)
doi: 10.3724/SP.J.1006.2016.01487 |
|
[20] | 何磊, 许利剑. 生物标记物在胃癌诊断及预后中作用的研究进展. 医学研究生学报, 2020, 33(9):1004-1008. |
HE L, XU L J. Research progress on the role of biomarkers in the diagnosis and prognosis of gastric cancer. Journal of Medical Postgraduates, 2020, 33(9):1004-1008. (in Chinese) | |
[21] | 潘晓晗, 郝春华, 陈芙蓉, 王维亭, 黄长江, 汤立达. 脑梗死生物标志物研究进展. 现代药物与临床, 2020, 35(1):189-196. |
PAN X H, HAO C H, CHEN F R, WANG W T, HUANG C J, TANG L D. Research progress on biomarkers for cerebral infraction. Drugs & Clinic, 2020, 35(1):189-196. (in Chinese) | |
[22] | 刘文娟, 常丽娟, 岳丽杰, 宋君, 张富丽, 王东, 吴佳蔚, 郭灵安, 雷绍荣. 两个玉米品种维管束鞘叶绿体的非光化学淬灭对干旱胁迫的响应. 中国农业科学, 2020, 53(8):1532-1544. |
LIU W J, CHANG L J, YUE L J, SONG J, ZHANG F L, WANG D, WU J W, GUO L A, LEI S R. Response of non-photochemical quenching in bundle sheath chloroplasts of two maize hybrids to drought stress. Scientia Agricultura Sinica, 2020, 53(8):1532-1544. (in Chinese) | |
[23] | 边大红, 刘梦星, 牛海峰, 魏钟博, 杜雄, 崔彦宏. 施氮时期对黄淮海平原夏玉米茎秆发育及倒伏的影响. 中国农业科学, 2017, 50(12):2294-2304. |
BIAN D H, LIU M X, NIU H F, WEI Z B, DU X, CUI Y H. Effects of nitrogen application times on stem traits and lodging of summer maize (Zea mays L.) in the Huang-huai-hai plain. Scientia Agricultura Sinica, 2017, 50(12):2294-2304. (in Chinese) | |
[24] | 周琦, 张富仓, 李志军, 强生才, 田建柯, 李国栋, 范军亮. 施氮时期对夏玉米生长、干物质转运与产量的影响. 干旱地区农业研究, 2018, 36(1):76-82. |
ZHOU Q, ZHANG F C, LI Z J, QIANG S C, TIAN J K, LI G D, FAN J L. Effects of nitrogen application at different stages on growth, yield, and dry matter transportation of summer maize. Agricultural Research in the Arid Areas, 2018, 36(1):76-82. (in Chinese) | |
[25] | 常程, 刘晶, 隋阳辉, 张书萍, 史磊, 肖万欣, 王和君, 王金艳, 徐亮. 施氮时期对春玉米果穗不同粒位籽粒发育及产量的影响. 辽宁农业科学, 2021, 2(2):8-12. |
CHANG C, LIU J, SUI Y H, ZHANG S P, SHI L, XIAO W X, WANG H J, WANG J Y, XU L. Effect of nitrogen application period on grain development in different ear positions and yield of spring maize. Liaoning Agricultural Sciences, 2021, 2(2):8-12. (in Chinese) | |
[26] | 宋建民, 田纪春, 赵世杰. 植物光合碳和氮代谢之间的关系及其调节. 植物生理学通讯, 1998, 34(3):230-238. |
SONG J M, TIAN J C, ZHAO S J. Relationship between photosynthetic carbon and nitrogen metabolism in plants and its regulation. Plant Physiology Communications, 1998, 34(3):230-238. (in Chinese) | |
[27] | 张凤路, 江亚丽, 赵国顺, 张俊花. 14C-同化物在玉米果穗上的分布与籽粒败育关系. 作物学报, 2006, 32(7):1104-1106. |
ZHANG F L, JIANG Y L, ZHAO G S, ZHANG J H. Relationship between distribution of 14C-assimilates and kernel abortion in maize. Acta Agronomica Sinica, 2006, 32(7):1104-1106. (in Chinese) | |
[28] | 汤继华, 谢惠玲, 黄绍敏, 胡彦民, 刘宗华, 季洪强, 寇志安. 缺氮条件下玉米自交系叶绿素含量与光合效率的变化. 华北农学报, 2005, 20(5):10-12. |
TANG J H, XIE H L, HUANG S M, HU Y M, LIU Z H, JI H Q, KOU Z A. The changes of chlorophyll content and photosynthetic productivity in maize inbred lines under the low-nitrogen stress. Acta Agriculturae Boreali-Sinica, 2005, 20(5):10-12. (in Chinese) | |
[29] | 吕丽华, 赵明, 赵久然, 陶洪斌, 王璞. 不同施氮量下夏玉米冠层结构及光合特性的变化. 中国农业科学, 2008, 41(9):2624-2632. |
LÜ L H, ZHAO M, ZHAO J R, TAO H B, WANG P. Canopy structure and photosynthesis of summer maize under different nitrogen fertilizer application rates. Scientia Agricultura Sinica, 2008, 41(9):2624-2632. (in Chinese) | |
[30] | LIU T B, KIM D W, NIITSU M, BERBERICH M, KUSANO T. POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of POLYAMINE OXIDASE 5. Plant Signaling & Behavior, 2014, 9(9):1559-2324. |
[31] |
DUBOS C, STRACKE R, GROTEWOLD E, WEISSHAAR B, MARTIN C, LEPINIEC L. MYB transcription factors in Arabidopsis. Trends in Plant Science, 2010, 15(10):573-581.
doi: 10.1016/j.tplants.2010.06.005 |
[32] |
STASWICK P E. Novel regulation of vegetative storage protein genes. The Plant Cell, 1990, 2(1):1-6.
doi: 10.2307/3869045 |
[33] |
GREENE B, WALKO R, HAKE S. Mutator insertions in an intron of the maize knotted1 gene result in dominant suppressible mutations. Genetics, 1994, 138(4):1275-1285.
doi: 10.1093/genetics/138.4.1275 |
[34] |
TSAI C Y, HUBER D M, GOVER D V. Relationship of N deposition to grain yield and N response of three maize hybrids. Crop Science, 1984, 24(2):277-281.
doi: 10.2135/cropsci1984.0011183X002400020016x |
[35] | 向春阳, 常强, 马兴林, 关义新, 凌碧莹, 张宝石. 玉米不同基因型对氮营养胁迫的反应. 黑龙江八一农垦大学学报, 2002, 14(4):5-7. |
XIANG C Y, CHANG Q, MA X L, GUAN Y X, LING B Y, ZHANG B S. Response to nitrogen stress on maize genotypes. Journal of Heilongjiang Bayi Agricultural University, 2002, 14(4):5-7. (in Chinese) |
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