Cloning and transformation of the CkCAD gene in Caragana korshinskii and analysis of its drought resistance function

doi:10.11686/cyxb2020150

Abstract

Abstract:

Drought stress seriously affects the growth and development and even survival status of plants， which is one of the main abiotic stress factors restricting the restoration of desert vegetation in northwest China. Previous studies in our laboratory have found that phenylpropanoid biosynthesis of Caragana korshinskii was the most significant metabolic pathway for differential expression as precipitation decreases following the Loess Plateau from south to north. This study focused on the lignin synthase gene CkCAD in the phenylpropanoid biosynthesis pathwayof C. korshinskii. A bioinformatics analysis showed that open reading frame of the CkCAD gene is 1074 bp， encoding 357 amino acids. Protein sequence alignment showed that CkCAD is closely related to Abrus precatorius， Medicago truncatula， Glycine max and Arachis hypogaea， with a similarity of more than 80%. It is most similar to the A. precatorius ApCAD. The protein is acidic and hydrophilic， with no transmembrane domain， and its subcellular location is in the cytoplasm. C. korshinskiiCkCAD was transferred into wild-type Arabidopsis thaliana with overexpression vector pCAMBIA1302 by the Agrobacterium-mediated method to obtain positive plants. The drought resistance was analyzed after screening and obtaining T₃ generation homozygous positive plants. It was found by Real-time fluorescence quantitative PCR and Western blot methods that CkCAD and its enzyme protein were stably expressed in T₃ Arabidopsis plants overexpressing CkCAD. Compared with wild type A. thaliana， the A. thaliana T₃ generation overexpressing CkCAD has longer vein length and greater vein density， shorter vein island length and shorter diameter， greater vein island density， more developed veins and higher lignin content. Simultaneous drought treatment revealed that the degree of leaf wilting， malondialdehyde content， and electrolyte leakage of overexpressed plants were lower than those of the wild type plants， while the relative water content was higher than that of the wild type plants. This study confirmed that the lignin synthase gene CkCAD of C. korshinskii can promote lignin synthesis and improve drought resistance of overexpressed A. thaliana plants under drought stress.

Key words: Caragana korshinskii, CkCAD, lignin, overexpression, drought resistance

Zhi-peng CHANG, Ying-ying SUN, Jia-yang LI, Chun-mei GONG. Cloning and transformation of the CkCAD gene in Caragana korshinskii and analysis of its drought resistance function[J]. Acta Prataculturae Sinica, 2021, 30(3): 68-80.

Figures/Tables 12

Table 1 Primers for C. korshinskiiCkCAD conversion and quantification in this study

引物名称Primer name	引物序列Primer sequence （5′-3′）
1302-CkCAD-F	GGACTCTTGACCATGATGGGTAGCCTTGAATCGG
1302-CkCAD-R	CTTCTCCTTTACTAGCTGATCAAGTTTACTGCCTTTGAC
Atactin-F	CACTACCGCAGAACGGGAAA
Atactin-R	GCGATGGCTGGAACAGAACC
CkCAD-F	TCTCTCCCCATACACCTACA
CkCAD-R	CCTTCTCTTTCCAAAACTCC
AtCAD-F	TAGAAGCAGGAGAAAAG
AtCAD-R	CAGGAACCATAGGATAA

Fig.1 Amplified bands of C. korshinskii CkCAD sequence

Fig.2 Phylogenetic tree and amino acid sequence comparison of C. korshinskii CkCAD and other species

Fig.3 CkCAD protein structure and subcellular localization prediction of C. korshinskii

Fig.4 Construction of pCAMBIA1302-35S::CkCAD overexpression vector

Fig.5 Screening of CkCAD transgenic Arabidopsis positive plants

Fig.6 C. korshinskiiCkCAD can be stably expressed in transgenic Arabidopsis

Fig.7 Growth phenotypes of CkCAD transformed Arabidopsis plants under drought stress

Fig.8 Variation trend of plant height and leaf area of wild-type and CkCAD transformed Arabidopsis plants under drought stress

Fig.9 Comparison of leaf vein phenotype between wild-type and CkCAD transgenic Arabidopsis plants

Fig.10 Analysis of vein characteristics of wild-type and CkCAD transgenic Arabidopsis plants

Fig.11 Comparison of drought resistance between wild-type and CkCAD-transformed Arabidopsis under drought stress

References 43

1	Gao L F. The expression of related genes CkERF2 and CkCOV1 in leaf vascular development during responding drought in Caragana korshinskii. Xianyang： Northwest A&F University， 2018.
	高丽芳. 叶脉发育相关基因CkERF2和CkCOV1在柠条叶脉响应干旱中的表达研究. 咸阳：西北农林科技大学， 2018.
2	Ning P B， Zhou Y L， Gao L F， et al. Unraveling the microRNA of Caragana korshinskii along a precipitation gradient on the Loess Plateau， China， using high-throughput sequencing. PLoS One， 2017， 12（2）： e0172017.
3	Li Y J， Zhao Z， Sun D X. Hydrological physiological characteristics of Caragana korshinskii under water stress. Journal of Northwest Forestry University， 2008， 23（3）： 1-4.
	李彦瑾，赵忠，孙德祥.干旱胁迫下柠条锦鸡儿的水分生理特征. 西北林学院报， 2008， 23（3）： 1-4.
4	Yao H， Zhao X Y， Li X M. Physiological response of 3 kinds of Caragana plant seedlings on continuous drought. Journal of Anhui Agricultural Sciences， 2009， 37（9）： 3915-3917.
	姚华，赵晓英，李晓梅. 3种锦鸡儿属植物幼苗对持续干旱的生理响应.安徽农业科学， 2009， 37（9）： 3915-3917.
5	Wang J J. The drought adaption of leaf functional traits in Caragana korshinskii KOM. under water gradient. Xianyang： Northwest A&F University， 2015.
	王佳佳. 水分梯度下柠条锦鸡儿叶功能属性的干旱适应性研究. 咸阳：西北农林科技大学， 2015.
6	Wei L L. Mechanism analysis of cold and drought tolerance of the transgenic Arabidopsis overexpressing DREB1 from Caragana korshinskii Kom. Hohhot： Inner Mongolia Agricultural University， 2013.
	魏丽丽. 过表达柠条锦鸡儿CkDREB1基因的拟南芥抗旱和抗冷的机理分析.呼和浩特：内蒙古农业大学， 2013.
7	Xia J X， Liu Y J， Yao S B， et al. Characterization and expression profiling of Camellia sinensis cinnamate 4-hydroxylase genes in phenylpropanoid pathways. Genes， 2017， 8（8）： 193.
8	Turner S R， Somerville C R. Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall. The Plant Cell， 1997， 9（5）： 689-701.
9	Xue C， Yao J L， Xue Y S， et al. PbrMYB169 positively regulates lignification of stone cells in pear fruit. Journal of Experimental Botany， 2019， 70（6）： 1801-1814.
10	Santos A B d， Bottcher A， Kiyota E， et al. Water stress alters lignin content and related gene expression in two sugarcane genotypes. Journal of Agricultural and Food Chemistry， 2015， 63（19）： 4708-4720.
11	Liu F R， Xie L F， Yao Z Y， et al. Equipment B： Caragana korshinskii phenylalanine ammonialyase is up-regulated in the phenylpropanoid biosynthesis pathway in response to drought stress. Biotechnology & Biotechnological Equipment， 2019， 33（1）： 842-854.
12	Cass C L， Peraldi A， Dowd P F， et al. Effects of phenylalanine ammonia lyase （PAL） knockdown on cell wall composition， biomass digestibility， and biotic and abiotic stress responses in Brachypodium. Journal of Experimental Botany， 2015， 66（14）： 4317-4335.
13	Sun Y Y. Study on drought response of genes related to lignin synthesis in Caragana korshinskii. Xianyang： Northwest A&F University， 2018.
	孙莹莹. 柠条锦鸡儿木质素合成基因的干旱响应研究. 咸阳：西北农林科技大学， 2018.
14	Clough S J， Bent A F. Floral dip： A simplified method for Agrobacterium‐mediated transformation of Arabidopsis thaliana. The Plant Journal， 1998， 16（6）： 735-743.
15	Xu C， Wang Y D， Yu Y C， et al. Degradation of MONOCULM 1 by APC/C TAD1 regulates rice tillering. Nature Communications， 2012， 3（1）： 1-9.
16	Zhang X H， Liu T J， Duan M M， et al. De novo transcriptome analysis of Sinapis alba in revealing the glucosinolate and phytochelatin pathways. Frontiers in Plant Science， 2016， 7： 259.
17	Ganguly D R， Crisp P A， Eichten S R， et al. The Arabidopsis DNA methylome is stable under transgenerational drought stress. Plant Physiology， 2017， 175（4）： 1893-1912.
18	Marshall D M， Muhaidat R， Brown N J， et al. Cleome， a genus closely related to Arabidopsis， contains species spanning a developmental progression from C₃ to C₄ photosynthesis. The Plant Journal， 2007， 51（5）： 886-896.
19	Hatfield R， Fukushima R S. Can lignin be accurately measured？ Crop Science， 2005， 45（3）： 832-839.
20	Kou X H， He Y L， Li Y F， et al. Effect of abscisic acid （ABA） and chitosan/nano-silica/sodium alginate composite film on the color development and quality of postharvest Chinese winter jujube （Zizyphus jujuba Mill. cv. Dongzao）. Food Chemistry， 2019， 270： 385-394.
21	Wang J B， Ding B， Guo Y L， et al. Overexpression of a wheat phospholipase D gene， TaPLDα， enhances tolerance to drought and osmotic stress in Arabidopsis thaliana. Planta， 2014， 240（1）： 103-115.
22	Zhou W F， Liu F R， Yao Z Y， et al. Growth adaptation characteristics of three Salsola species with different photosynthetic systems. Acta Prataculturae Sinica， 2019， 28（10）： 78-90.
	周文菲，刘芙蓉，姚甄业，等. 猪毛菜属 3 种不同光合型物种的生长适应特征比较. 草业学报， 2019， 28（10）： 78-90.
23	Tang H M， Liu S Z， Hill‐Skinner S， et al. The maize brown midrib2 （bm2） gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation. The Plant Journal， 2014， 77（3）： 380-392.
24	Wang J H， Feng J J， Jia W T， et al. Genome-wide identification of sorghum bicolor laccases reveals potential targets for lignin modification. Frontiers in Plant Science， 2017， 8： 714.
25	Mottiar Y， Vanholme R， Boerjan W， et al. Designer lignins： Harnessing the plasticity of lignification. Current Opinion in Biotechnology， 2016， 37： 190-200.
26	Wang P， Dudareva N， Morgan J A， et al. Genetic manipulation of lignocellulosic biomass for bioenergy. Current Opinion in Chemical Biology， 2015， 29： 32-39.
27	Coleman H D， Park J Y， Nair R， et al. RNAi-mediated suppression of p-coumaroyl-CoA 3′-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism. Proceedings of the National Academy of Sciences， 2008， 105（11）： 4501-4506.
28	Amrhein N， Frank G， Lemm G， et al. Inhibition of lignin formation by L-alpha-aminooxy-beta-phenylpropionic acid， an inhibitor of phenylalanine ammonia-lyase. European Journal of Cell Biology， 1983， 29（2）： 139-144.
29	Smart C C， Amrhein N. The influence of lignification on the development of vascular tissue in Vigna radiata L. Protoplasma， 1985， 124（1/2）： 87-95.
30	Li Z， Peng Y， Ma X. Different response on drought tolerance and post-drought recovery between the small-leafed and the large-leafed white clover （Trifolium repens L.） associated with antioxidative enzyme protection and lignin metabolism. Acta Physiologiae Plantarum， 2013， 35（1）： 213-222.
31	Cheng X， Li G， Ma C， et al. Comprehensive genome-wide analysis of the pear （Pyrus bretschneideri） laccase gene （PbLAC） family and functional identification of PbLAC1 involved in lignin biosynthesis. PLoS One， 2019， 14： e0210892.
32	Zhong R， Taylor J J， Ye Z H. Disruption of interfascicular fiber differentiation in an Arabidopsis mutant. The Plant Cell， 1997， 9（12）： 2159-2170.
33	Lens F， Tixier A， Cochard H， et al. Embolism resistance as a key mechanism to understand adaptive plant strategies. Current Opinion in Plant Biology， 2013， 16（3）： 287-292.
34	Adams H D， Zeppel M J， Anderegg W R， et al. A multi-species synthesis of physiological mechanisms in drought-induced tree mortality. Nature Ecology & Evolution， 2017， 1（9）： 1285-1291.
35	Anderegg W R， Klein T， Bartlett M， et al. Meta-analysis reveals that hydraulic traits explain cross-species patterns of drought-induced tree mortality across the globe. Proceedings of the National Academy of Sciences， 2016， 113（18）： 5024-5029.
36	Pereira L， Domingues-Junior A P， Jansen S， et al. Is embolism resistance in plant xylem associated with quantity and characteristics of lignin？ Trees， 2017（33）： 1-10.
37	Jansen S， Choat B， Pletsers A. Morphological variation of intervessel pit membranes and implications to xylem function in angiosperms. American Journal of Botany， 2009， 96（2）： 409-419.
38	Li S， Lens F， Espino S， et al. Intervessel pit membrane thickness as a key determinant of embolism resistance in angiosperm xylem. Iawa Journal， 2016， 37（2）： 152-171.
39	Dória L C， Podadera D S， del Arco M， et al. Insular woody daisies （Argyranthemum， Asteraceae） are more resistant to drought‐induced hydraulic failure than their herbaceous relatives. Functional Ecology， 2018， 32（6）： 1467-1478.
40	Dória L C， Meijs C， Podadera D S， et al. Embolism resistance in stems of herbaceous Brassicaceae and Asteraceae is linked to differences in woodiness and precipitation. Annals of Botany， 2018， 20： 1-13.
41	Xu T， Zhao C Z， Han L， et al. Correlation between vein density and water use efficiency of Salix matsudana in Zhangye wetland， China. Chinese Journal of Plant Ecology， 2017， 41（7）： 761-769.
	徐婷，赵成章，韩玲，等. 张掖湿地旱柳叶脉密度与水分利用效率的关系. 植物生态学报， 2017， 41（7）： 761-769.
42	Sack L， Scoffoni C. Leaf venation： Structure， function， development， evolution， ecology and applications in the past， present and future. New Phytologist， 2013， 198（4）： 983-1000.
43	Nardini A， Raimondo F， Lo Gullo M A， et al. Leafminers help us understand leaf hydraulic design. Plant， Cell & Environment， 2010， 33（7）： 1091-1100.

[1]	Hai-feng HE, Cheng-hong YAN, Na WU, Ji-li LIU, Yu-han JIA. Effects of different nitrogen levels on photosynthetic characteristics and drought resistance of switchgrass （Panicum virgatum） [J]. Acta Prataculturae Sinica, 2021, 30(1): 107-115.
[2]	ZENG Ling-shuang, LI Pei-ying, SUN Xiao-fan, SUN Zong-jiu. A multi-trait evaluation of drought resistance of bermudagrass (Cynodon dactylon) germplasm from different habitats in Xinjiang province [J]. Acta Prataculturae Sinica, 2020, 29(8): 155-169.
[3]	ZHANG Xue-ting, WANG Xin-yong, YANG Wen-xiong, LIU Na, YANG Chang-gang. Evaluation of water-saving and drought-resistant maize varieties in the Hexi oasis irrigation corridor [J]. Acta Prataculturae Sinica, 2020, 29(2): 134-148.
[4]	Hai-tao CHANG, Ren-tao LIU, Wei CHEN, An-ning ZHANG, Xiao-an ZUO. Distribution of ground-active arthropod community structure after introduction of Caragana korshinskii into Reaumuria soongorica shrubland on the Urat desert steppe， Inner Mongolia [J]. Acta Prataculturae Sinica, 2020, 29(12): 188-197.
[5]	YANG Liu-hui, YIN Hang, HUANG Qin-mei, ZHANG Yan-ni, HE Miao, ZHOU Yun-wei. An analysis of the response of the LpWRKY20 gene to abiotic stress and its role in drought resistance [J]. Acta Prataculturae Sinica, 2020, 29(1): 193-202.
[6]	WU Juan-zi, QIAN Chen, LIU Zhi-wei, PAN Yu-mei, ZHONG Xiao-xian. De novo transcriptomic analysis for lignin synthesis in Cenchrus purpureus using RNA-seq [J]. Acta Prataculturae Sinica, 2019, 28(1): 150-161.
[7]	GUO Xing, XIE Fei, YAN Qian-qian, CAO Xiu-wen, YANG Fan. Effect of fulvic acid on drought resistance of nursery stocks in a dry valley of the Bailongjiang River, Gansu [J]. Acta Prataculturae Sinica, 2018, 27(8): 86-94.
[8]	LIU Ting-ting, CHEN Dao-qian, WANG Shi-wen, YIN Li-na, DENG Xi-ping. Physio-ecological responses to drought and subsequent re-watering in sorghum seedlings [J]. Acta Prataculturae Sinica, 2018, 27(6): 100-110.
[9]	XU Pian-pian, WANG Jian-zhu. Drought resistance of three common slope plants determined in a simulated drought experiment [J]. Acta Prataculturae Sinica, 2018, 27(2): 36-47.
[10]	SHI Jing-Ang, ZHANG Bing, XIAO Xiao-Lin, MA Jing-Jing, YANG Xiang-Yang, LIU Jian-Xiu. Genome-wide identification and characterization of the cinnamyl alcohol dehydrogenase gene family in Zoysia japonica [J]. Acta Prataculturae Sinica, 2017, 26(6): 111-119.
[11]	FAN Zhi-Xia, LI Shao-Cai, SUN Hai-Long. Physiological response of Amorpha fruiticosa to drought stress under paclobutrazol application and an evaluation of drought resistance [J]. Acta Prataculturae Sinica, 2017, 26(3): 132-141.
[12]	ZHAO Jing-Zhong, KONG Dong-Sheng, WANG Li, GUO You-Yan. Effects of low temperature stratification on emergence of Lycium ruthenicum under drought at different sowing depths [J]. Acta Prataculturae Sinica, 2017, 26(12): 56-66.
[13]	WU Mei-Yan, HAO Ruo-Chao, ZHANG Wen-Ying. Effects of Piriformospora indica fungus on growth and drought resistance in alfalfa under water deficit stress [J]. Acta Prataculturae Sinica, 2016, 25(5): 78-86.
[14]	LIU Ke-Biao, JIANG Sheng-Xiu. Responses of Apoceynum venetum seed germination to drought and salt stress [J]. Acta Prataculturae Sinica, 2016, 25(5): 214-222.
[15]	WANG Yi, GUO Hai-Lin, CHEN Jing-Bo, ZONG Jun-Qin, LI Dan-Dan, JIANG Yi-Wei, LIU Jian-Xiu. Preliminary evaluation of drought resistance for the hybrid zoysiagrass ‘Suzhi No.1’ [J]. Acta Prataculturae Sinica, 2016, 25(5): 30-39.