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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (11): 2966-2977.doi: 10.3724/SP.J.1006.2023.21063

所属专题： 小麦：遗传育种·种质资源·分子遗传学

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

不同氮利用效率小麦品种TaNRT/TaNPF家族基因表达特点

王露露1(), 仪子博1, 王浩哲1, 能芙蓉2, 马新明1, 张志勇1,*(), 王小纯1,2,*()   

  1. 1省部共建小麦玉米作物学国家重点实验室 / 河南农业大学, 河南郑州45000
    2河南农业大学生命科学学院, 河南郑州450002
  • 收稿日期:2022-09-13 接受日期:2023-04-17 出版日期:2023-11-12 网络出版日期:2023-05-16
  • 通讯作者: 王小纯, E-mail: xiaochun.w@163.com; 张志勇, E-mail: zhiyongzhang@henau.edu.cn
  • 作者简介:E-mail: wanglulu9501@163.com
  • 基金资助:
    国家自然科学基金项目(32071956)

Gene expression characteristics of TaNRT/TaNPF family in wheat cultivars with different nitrogen efficiency

WANG Lu-Lu1(), YI Zi-Bo1, WANG Hao-Zhe1, NAI Fu-Rong2, MA Xin-Ming1, ZHANG Zhi-Yong1,*(), WANG Xiao-Chun1,2,*()   

  1. 1Co-constuction State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2Department of Biochemistry, College of Life Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2022-09-13 Accepted:2023-04-17 Published:2023-11-12 Published online:2023-05-16
  • Contact: 王小纯, E-mail: xiaochun.w@163.com; 张志勇, E-mail: zhiyongzhang@henau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32071956)

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摘要：

氮素是小麦生长发育的必需元素之一, NO3--N是小麦从土壤中获取氮的主要形式。NRT/NPF家族基因编码膜转运蛋白, 主要参与植物NO3--N吸收、运输及分配。为了解小麦NRT/NPF家族基因与氮素利用的关系, 选用氮高效小麦品种周麦27 (ZM27)和氮低效品种矮抗58 (AK58), 利用二代测序技术, 研究了不同氮水平(N120、N225、N330)开花期TaNRT/TaNPF家族基因在旗叶中的表达特点。结果表明, 二代测序鉴定到386个TaNRT/TaNPF家族基因; 与AK58相比, ZM27在氮减量(N120)、正常(N225)、过量(N330)条件下差异表达基因分别为27、16和23个, 上调表达基因分别为16 (59.26%)、12 (75%)和19 (82.61%)个; 减氮条件下ZM27有7个特异下调表达基因, 氮过量条件下TaNPF8.1表达量最高, 且显著上调1.5倍。可见, TaNRT/TaNPF家族基因表达水平受施氮量及品种调控。利用小麦网络数据库分析发现TaNRT/TaNPF家族基因表达具有组织特异性及染色体偏好性, 旗叶表达量最高的TaNPF8.1定位于3A染色体, 根系特异表达的TaNRT2.2和TaNRT3.1主要分布于6号染色体, 茎秆特异表达的TaNPF4.5主要分布在2号染色体。qRT-PCR分析显示TaNRT/TaNPF基因表达特点与二代转录组及网络数据结果一致。TaNPF8.1、TaNPF4.5和TaNRT3.1蛋白互作分析发现, NO3--N转运可能还需要转录因子MYB、叶绿素A-B结合蛋白、伴侣蛋白等协同参与, 为进一步研究TaNRT/TaNPF家族表达与氮素吸收利用的关系奠定了基础。

关键词: 小麦, NRT/NPF, 氮效率, 差异表达, 组织特异性

Abstract:

Nitrogen is one of the essential elements for wheat growth and development, and NO3--N is the main form of nitrogen that wheat obtains from soil. NRT/NPF genes family encode membrane transporters, which are mainly involved in NO3--N absorption, transport, and allocation in plants. In order to understand the relationship between NRT/NPF family and nitrogen utilization in wheat, the relative expression characteristic of TaNRT/TaNPF family in flag leaves of N-efficient wheat cultivars Zhoumai 27 (ZM27) and N-inefficient wheat cultivars Aikang 58 (AK58) at flowering stage were studied with the second-generation sequencing technology. The results showed that 386 genes of TaNRT/TaNPF family were identified in the second generation transcriptome database. Compared with AK58, there were 27, 16, and 23 differentially expressed genes in ZM27 in reducing (N120), normal (N225), and excessive (N330) nitrogen treatments. There were 16 (59.26%), 12 (75%), and 19 (82.61%) up-regulated genes in ZM27, respectively. Seven genes were down-regulated in ZM27 in reducing nitrogen treatment. The relative expression level of TaNPF8.1 was the highest and significantly up-regulated by 1.5 times in nitrogen excessive condition. In conclusion, the relative expression of TaNRT/TaNPF family genes was regulated by nitrogen application rate and cultivar. Wheat network database showed that the relative expression of TaNRT/TaNPF family had tissue specificity and chromosomal preference. The highest expression level of TaNPF8.1 in flag leaf was located on chromosome 3A, and the root specific expression TaNRT2.2 and TaNRT3.1 were mainly distributed on chromosome 6. The stem specific expression of TaNPF4.5 was mainly distributed on chromosome 2. The qRT-PCR of TaNRT/TaNPF genes were consistent with the results of the second-generation transcriptome and network data. Interaction analysis of TaNPF8.1, TaNPF4.5, and TaNRT3.1 revealed that NO3--N transport may also require the collaborative participation of transcription factor MYB, chlorophyll A-B binding protein, and chaperone protein. These findings laid a foundation for further studies on the relationship between TaNRT/TaNPF family expression and nitrogen uptake and utilization.

Key words: wheat, NRT/NPF, nitrogen use efficiency, different expression, tissue specificity

表1

qRT-PCR引物序列"

基因名称
Gene name		 正向引物序列
Forward sequence (5′-3′)		 反向引物序列
Reverse sequence (5′-3′)
NRT2.2 CCTTGGTATCATCTCCGGGC GAGGGTGACAGGAAGAGTGC
NRT3.1 CCCCCAGCTCTTCTCTTGC TCACCGGCAGCTTGGAGA
NPF4.5 AGCAGCAACCTACAAGCAGT TGGAAGCTCTCACTCCTCCA
NPF5.102D GGAAACCACGCACAGAAGC CGTGGGTGTTTTGTTCCTGTC
NPF5.101A CCCTCGTCCTGTAGCTTGAC CCAGCAGAGGACGCCATTAA
NPF8.13A TCAGAGAACGCAACCACTGT CCTGCAGGCCTTTTGATTCG
NPF8.34A ATTCCTTCTCCCTCCCCGAA GCTGGGATACTCTGCTCGGA
NPF8.34D CCCTTCCCCTTGCTTGCTTA TCGGGGTGGGAGTTGAGTTA
NPF8.31B AACGTGAAAGGGGGAGGGAT GGTTTGCCGTTGTGATTTGC
CHS2 CACCACAATAATCCTTGCTA ACTTATGACCCAATTACTGAAT
PTR1 AAGCGGGCTGATGGTGATGG CGGCGGTGACCAGGTAGAAC
POD70 CTGAGGCGAACAGCGACCTC CGGGCTCAGCTGCTTCTTGT
RPP13 TGGGTTCGGTGGCATAGGGA CCCAAGCCCGTGCTTCGTAT
ATPase CGAGGCCACCAATGACG AGTATGGTTTCAAGAAGGCGTC
TEF TCGTGGTCATTGGCCACG CAGCACAGTCAGCCTGGGAG

图1

TaNRT/TaNPF家族基因差异表达分析 A: 数据来自网络数据库(https://www.wheatproteome.org/), 蓝-白-红表示基因表达量由低到高; B: 数据来自二代转录组测序。"

图2

不同品种不同氮水平下TaNRT/TaNPF家族基因在旗叶中差异表达特点 A: 不同品种不同氮水平下TaNRT/TaNPF家族在旗叶中的差异基因数, 红箭头: 上调基因, 绿箭头: 下调基因; B: 不同氮水平下TaNRT/TaNPF上调表达基因数; C: 不同氮水平下TaNRT/TaNPF下调表达基因数."

附表1

不同氮效率小麦品种差异基因表达量分析"

类型
Type		 基因名称
Gene name		 AK58 N120 AK58 N225 AK58 N330 ZM27 N120 ZM27 N225 ZM27 N330
Up-regulated DEGs NPF2.3 TraesCS4A03G1098900 0.14 0.29 0.03 0.69 0.35 0.50
NPF2.11 TraesCS5A03G0008900 2.79 3.03 2.19 5.70 5.64 5.08
TraesCS5B03G0001600 2.39 2.12 2.06 5.86 6.84 5.24
TraesCS5B03G0102800 8.11 6.37 6.52 11.29 11.94 9.35
NPF4.4 TraesCS4A03G0599700 0.28 0.41 0.36 0.73 0.75 1.01
NPF5.1 TraesCS7A03G1119900 2.33 2.78 2.50 3.05 3.92 3.64
TraesCS7D03G1067400 0.50 0.82 0.56 0.64 0.79 1.05
NPF5.8 TraesCS7B03G0265800 0.28 0.39 0.27 0.56 0.50 0.56
TraesCS7D03G0440800 0.60 1.17 0.91 1.28 1.37 1.45
NPF5.10 TraesCS3A03G0903800 0.59 0.75 0.43 0.71 1.01 0.93
NPF5.13 TraesCS2D03G1323700 0.88 1.19 1.23 1.11 1.58 1.93
NPF6.2 TraesCS1B03G0077900 0.33 0.39 0.37 0.19 0.23 0.95
NPF7.3 TraesCS6D03G0593500 0.21 0.29 0.16 0.44 0.37 0.40
NPF8.1 TraesCS3A03G0922900 35.44 37.45 34.96 51.56 52.52 53.18
TraesCS3A03G0923200 3.28 3.04 3.17 5.67 6.21 5.36
TraesCS3D03G0852600 0.82 0.77 0.62 0.72 0.63 0.96
TriticumnewGene_8087 17.36 16.63 15.86 25.28 25.27 24.94
NPF8.3 TraesCS7A03G1290100 0.09 0.13 0.58 1.02 0.54 1.13
TraesCS7B03G0835300 1.98 1.99 1.65 2.60 2.77 2.73
Down-regulated DEGs NRT2.4 TraesCS7B03G0882400 5.31 4.37 4.39 0.19 0.09 0.25
NPF2.11 TraesCS5D03G0010900 2.53 1.53 1.67 1.49 1.42 1.41
NPF4.6 TraesCS5D03G0163200 2.17 1.20 1.82 1.29 0.50 0.44
NPF5.2 TraesCS4D03G0779400 1.79 1.32 1.56 1.07 0.98 1.37
NPF5.10 TraesCS2A03G1350700 1.89 2.30 2.51 0.99 1.33 1.26
TraesCS2B03G1540700 1.55 1.39 1.80 0.94 1.09 1.21
TraesCS3A03G0902900 0.93 0.83 0.89 0.52 0.72 0.64
NPF8.3 TraesCS6B03G1146900 1.10 0.98 1.38 0.54 0.79 0.92
TraesCS7B03G0839000 1.56 1.53 2.33 0.24 0.43 0.56
TraesCS7B03G0839200 7.12 7.14 6.78 4.81 5.00 4.94
NPF8.5 TraesCS2D03G0016800 2.02 1.59 1.79 1.08 1.52 1.51

图3

qRT-PCR验证转录组数据 A: 所选差异表达基因qRT-PCR检测; B: 所选基因对应的FPKM值。矮抗58: AK58; 周麦27: ZM27。N120: 减氮120 kg hm-2; N225: 正常氮225 kg hm-2; N330: 过量氮330 kg hm-2; NPF5.10-1A、NPF5.10-2D、NPF8.1-3A、NPF8.3-1B、NPF8.3-4A、NPF8.3-4D: 硝酸转运蛋白; CHS2: 查耳酮合成酶2; PR1: 致病相关蛋白1; POD70: 过氧化物酶70; RPP13: 抗病蛋白。"

图4

品种及氮水平对不同小麦组织TaNRT/TaNPF家族基因表达的影响 蓝-白-红表示基因表达量由低到高。TaNPF8.1、TaNPF4.5、TaNRT2.2、TaNRT3.1: 硝酸转运蛋白; Root: 根系; Peduncle:穂下节; Leaf: 旗叶; Sheat: 旗叶鞘; Grain: 籽粒; 缩写同图3。"

图5

TaNRT/TaNPF家族差异基因功能预测 蓝-白-红表示基因表达量由低到高。"

图6

TaNRT/TaNPF家族基因染色体定位 A: 组织特异表达TaNRT/TaNPF基因染色体定位; B: 氮调控差异表达TaNRT/TaNPF基因染色体定位。"

图7

组织特异表达的TaNRT/TaNPF的蛋白互作网络 蛋白质用节点表示, 相互作用用连线表示. 灰色: 转录因子; 黄色: 无机离子转运及代谢蛋白; 草绿色: 天冬氨酸蛋白酶; 红色: 叶绿素A-B结合蛋白; 紫色: 蛋白酶抑制剂; 绿色: 藏花酸葡糖基转移酶; 黑色: GPI-anchored蛋白; 蓝色: 花青素; 宝石蓝: 伴侣蛋白; 玫红色: 烷烃羟化酶; 浅黄色: 烟酰胺腺嘌呤二核苷酸激酶; 浅绿色: 高亲和力硝酸转运蛋白; 浅粉色: 糖基转移酶。"

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