FSHW | Transcriptomic studies of different varieties and developmental stages: Mining key genes for oil and fatty acid biosynthesis in three Xinjiang characteristic walnuts

Introduction

Walnut ( Juglans regia.

) is a good source of lipids and polyunsaturated fatty acids (PUFA.

Asia, North America, Europe and South America are the four major producing regions of walnut in the wor.

China is one of the important origins of walnuts and one of the main producers of walnuts in the wor.

The walnut producing areas in China are widely distributed and can be divided into four main cultivation areas: northwest, east coast, central China and southwe.

With the rapid development of the walnut industry, China's walnut output has ranked first in the world in recent years, and has cultivated abundant and high-quality germplasm resourc.

Xinjiang is one of the most important producing areas in Chi.

In 2016, the total output of walnuts reached 692,800 tons, ranking second in the country, second only to Yunn.

Aksu, Hotan and Kashgar in southern Xinjiang are the three major cultivation areas in Xinjia.

The walnut industry in southern Xinjiang has become a local pillar industry, and walnut income accounts for almost half of farmers' inco.

In recent years, with the improvement of the degree of mechanization and the improvement of breeding technology, the unit output of Xinjiang walnut has continued to increase, and it has become the second largest tree species in the characteristic forest fruit industry after juju.

However, there is still a big gap between China and the advanced countries in the wor.

For example, although the walnut production and planting area in the United States are far lower than those in China, due to the highest production level and the degree of improved varieties, the walnut market share and export share are both in the 50% or .

As one of the important edible nuts, walnuts are rich in protein, amino acids, fats, carbohydrates and various trace elements and minera.

Ripe walnut kernels contain 61%-74% oil, 15%-19% protein, 8%-13% total carbohydrates, and 3%-4% moistu.

Fatty acids are the main nutritional components of walnut kernels, of which unsaturated fatty acids account for 90%, including two essential fatty acids for the human body, namely linoleic acid and α -linolenic acid, and the ratio of the two is between 4:1 and 6: It is believed to be effective in preventing cardiovascular disea.

Due to its rich nutritional value and bioactive components, walnuts have many functions on the human body, such as brain-enhancing and intelligence, lowering blood lipids, lowering blood sugar, anti-oxidation and anti-cancer, so they are widely used in nutritional functional foo.

Most of the previous studies on walnut oil focused on the comparison of oil and fatty acid composition in different walnut varieties and the change rules at different developmental stages, but the molecular mechanism of oil accumulation was rarely report.

Xinjiang has unique climatic and topographical conditions, and is one of the most suitable regions in the world to grow walnu.

With the release of walnut genome data, related genes involved in walnut oil metabolism have been discover.

However, key genes for walnut triglyceride (TAG) assembly and transcriptional regulators related to walnut lipid synthesis have not been identified in previous related studi.

In addition, there is a lack of research on the oil content and fatty acid composition of different varieties, the key genes involved and the possible differences in expression patter.

The team of Professor Shen Lirong from the Department of Food Science and Nutrition of Zhejiang University and the team of Professor Wu Cuiyun from the School of Landscape Architecture of Tarim University conducted a transcriptomic, lipid and fatty acid comparative analysis of three Xinjiang characteristic walnuts and their different developmental stag.

The potential molecular mechanism of unsaturated fatty acid (PUFA) synthesis and the key genes involved in the lipid synthesis pathway of different varieties were excavated, which provided a theoretical basis for molecular breeding of Xinjiang characteristic walnut, increasing the oil content of walnut and improving the composition of PU.

The first author of this article is Wang Wenqiang, a postgraduate student in the Department of Food Science and Nutrition, Zhejiang Universi.

Graphical abstract

Analysis of oil content and fatty acid composition of different walnut varieties during oil accumulation period

The oil content and fatty acid composition of three walnut cultivars "Zhipi walnut (ZP)", "Xinwu 417 (W417)" and "Xinwen 81 (W81)" at different developmental stages were determin.

The results showed that the oil content of walnuts of the three varieties showed a "slow-fast-slow" variation patte.

The oil content of mature walnuts was close to 70% (Figure 1), and the proportion of unsaturated fatty acids reached more than 9
Walnut oil contains 6 kinds of fatty acids, including palmitic acid, stearic acid, oleic acid, linoleic acid, α -linolenic acid and eicosenoic acid, among which linoleic acid and oleic acid have the highest content, showing a synchronous change of decreasing and increasing respective.

trend (F.

2), it is speculated that there is a certain mutual transformation relationship between linoleic acid and oleic acid during the development of waln.

According to the change rules of oil content and fatty acid composition, this study determined that the three developmental periods of post-anthesis 84 d (T1), 98 d (T2) and 119 d (T3) were suitable for transcriptomic analys.

(A) Morphological characteristics of three walnut cultivars 84, 98 and 119 DAF during embryo development; (B) Dynamic changes of total oil content during embryo development of three walnut cultiva.

F.

1 Profile and total oil content of three walnut varieties (ZP, W417 and W81)

F.

2 Variation trend of fatty acid composition during embryonic development of three walnut cultivars (ZP, W417 and W81)

Transcriptome sequencing, assembly and gene function annotation of three walnut cultivars at different developmental stages

Transcriptome sequencing of 27 walnut samples derived from 3 walnut varieties at 3 developmental stages yielded a total of 666 Gb, 721 Gb, and 764 Gb Rawbases, excluding adaptors, in the sequencing of W417, W81, and ZP, respectively After sequencing, low-quality bases and indeterminate bases generated more than 97% of the total sequencing data volume (Cleanbase.

All 34 112 expressed genes were annotated to 6 public databases of NR, Swiss-Prot, Pfam, COG, GO and KEGG, and a total of 30 320 (888%) expressed genes were annotated in 6 databases, among which COG and NR The most annotated genes in the database were 30 312 (886%) and 29 862 (854%), respective.

While the GO and KEGG databases had the lowest annotation rates, annotating 15 279 (479%) and 12 992 (309%) genes, respectively (Table

Table 1 Unigene functional annotation results

PCA analysis and differential gene expression analysis among walnut samples

To understand the dynamic changes of gene expression during nuclear development, differentially expressed genes (DEGs) analysis was performed using transcripts per million (TPM) data from 27 samples (F.

3.

Three pairwise comparison groups from different developmental stages and breeds were analyz.

The control groups (T1 vs T2, T2 vs T3 and T1 vs T3) at three developmental stages W417, W81 and ZP contained 1 800, 1 145 and 1 352 DEGs, respectively, resulting in a total of 3 375 non-redundant DEGs (F.

3.

.

There were 12 094, 8 133 and 7 402 non-redundant DEGs in ZP and W417, ZP and W81, and W417 and W81 in three stages, respective.

In this way, 2 578 differentially co-expressed DEGs were included in the three comparisons (F.

3.

A total of 5 040 non-redundant DEGs redundant genes were identified, and 913 DEGs were found from different periods and varieties (F.

3.

(A) Principal component analysis of 27 walnut samples; (B) the number of DEGS intersections between different stages of W417, W81 and ZP; (C) the number of DEGs binding between W417, W81 and ZP in three developmental stages; (D) The sum of the DEGs of the union of B and the intersection o.

F.

3 Principal component analysis and Venn diagram of differential genes (DEGs) of three walnut varieties

Cluster analysis of differentially expressed genes

To better understand the expression patterns of differential genes between groups, cluster analysis was perform.

Based on the TPM value, cluster analysis was performed on 5 040 DEGs co-expressed in different periods and varieti.

The results indicated that the T1 and T2 phases of the 3 cultivars were well clustered and that genes in subcluster IV and subcluster V may play key roles in lipid biosynthesis and nuclear development (F.

(A) Hierarchical clustering analysis of differentially expressed genes, the color bar represents the expression level of differentially expressed genes after normalization to lg TPM, red represents high expression, blue means low expression; (B) 5 subgroups of differentially expressed genes expression mo.

Figure 4 Cluster analysis of differentially expressed genes

GO functional annotation and KEGG enrichment analysis of differentially expressed genes

To determine the specific functions of these differentially expressed genes, GO functional annotation analysis was perform.

The identified differentially expressed genes (3 375 and 2 578, respectively) between samples of different breeds and different developmental stages were annotated into the GO database, and 1 348 genes (Group I) and 1 795 genes (Group I) were annotated respectively ( Group I.

These two groups of genes were divided into three main functional categories, including biological process (BP), cellular component (CC) and molecular function (MF), and were further divided into 36 genes containing 10 subclasses of more than one ge.

Among them, the BP class is divided into 13 sub-classes, the CC class is divided into 12 sub-classes, and the MF class is mapped to 11 GO ter.

The annotation results of these genes showed that different varieties of walnut undergo strong metabolic activities during growth and development (F.

Figure 5 GO functional classification of differentially expressed genes in different breeds (Group I) and differentially expressed genes in different developmental stages (Group II)

The difference between each developmental stage was 3 375 DE.

Through KEGG enrichment analysis, 2 578 DEGs among varieties were analyzed to explore the metabolic pathways involved in these DEGs (F.

(A) KEGG enrichment analysis diagram of differentially expressed genes in Group III; (B) KEGG enrichment analysis diagram of differentially expressed genes in Group .

Figure 6 KEGG enrichment analysis of differentially expressed genes

Identification of genes related to lipid synthesis

Mining differentially expressed genes involved in oil biosynthesis is crucial to explore the molecular mechanism of oil accumulation in waln.

Therefore, a lipid biosynthesis pathway visualized by schematic diagram and heatmap was constructed to show the dynamic changes of gene expression levels during embryonic development in three walnut varieties, including 64 genes for fatty acid (FA) de novo synthesis and 45 genes for de novo fatty acid (FA) synthes.

For triacylglycerol assembly, 17 genes for lipid biosynthesis in oil bodies, 10 key enzymes (ACCase, LACS6, LACS8, SAD, FAD2, FAD3, LPAAT1, DGAT2, PDAT2, and PLC) encoded by 19 genes It is highly related to lipid biosynthesis, among which SAD, FAD2 and FAD3 are highly expressed in walnut kernel, which may be the main reason why walnut is rich in unsaturated fatty aci.

Then, through the correlation analysis of the expression levels of 27 walnut samples, five transcriptional regulators related to oil synthesis, including WRI1, ABI3, FUS3, PKL and VAL1, were identifi.

The main regulatory genes of these transcription factors include ACCase, KASⅡ, LACS, FAD3 and LPAAT (Figure

The color of the heatmap represents the logarithm of the fold difference of genes between different samples, red represents up-regulation and blue represents down-regulati.

Figure 7 Differentially expressed genes in the fat synthesis pathway in walnut

To further investigate the transcriptional level correlations between the identified transcription factors and genes involved in lipid biosynthesis, the correlation coefficients calculated using the Person algorithm were visualized with a heat map (F.

The color of the heatmap represents the magnitude of the correlation coefficient, red represents positive correlation, and blue represents negative correlati.

Turquoise indicates that the absolute value of the correlation coefficient between genes is less than 5, and q > 0

Figure 8 Correlation analysis between transcription factors and the expression levels of genes related to lipid and lipid synthesis

Finally, the expression of 9 key genes in ZP was verified by qRT-PCR (5 genes were verified by W417 and W81 respectively), which proved that there was a strong correlation between the quantitative results and the transcriptome sequencing data ( R ² =90889, P =62× ^10-32.

These findings will provide comprehensive understanding and valuable information for walnut genetic engineering and molecular breedi.

In this study, the transcriptomic, lipid and molecular analysis of three Xinjiang characteristic walnuts, namely "Paper-skin walnut (ZP)", "Xinwu 417 (W417)", "Xinwen 81 (W81)" and their different developmental stages were carried o.

Comparative analysis of fatty aci.

Lipid analysis results showed that the oil content of mature walnut kernels was close to 70%, of which more than 90% of the fatty acids were PUF.

Transcriptomic analysis identified 126 candidate genes, including 64 genes for fatty acid de novo synthesis, 45 genes for triacylglycerol assembly, and 17 genes for lipid biosynthesis in oil bodies; 19 genes encoded ACCase, LACS6 , LACS8, SAD, FAD2, FAD3, LPAAT1, DGAT2, PDAT2, PLC and other 10 key enzymes that are highly related to lipid biosynthesis; FUS3, PKL and VAL1 are five transcriptional regulators related to lipid synthesis, and it is determined that their main regulatory genes may include ACCase, KASII, LACS, FAD3 and LPA.

These findings will provide comprehensive understanding and valuable information for walnut genetic engineering and molecular breedi.

Wang Wenqiang , graduated from the School of Engineering and Food Science of Zhejiang University with a master's degree, his main research direction is food nutrition and safe.

Shen Lirong is a professor at the School of Biosystems Engineering and Food Science, Zhejiang University, a visiting scholar (postdoctoral fellow) at the Center for Human Aging Research at Tufts University, Harvard Medical School and the Ministry of Agricultu.

He is currently a professor at the Department of Food Science and Nutrition at Zhejiang Universi.

Academic part-time jobs include members of American Nutrition Society, Asia-Pacific Clinical Nutrition Society, and Asia-Pacific Vegetarian Alliance; executive director of Nutritional Translational Medicine Branch of Chinese Nutrition Society, expert of Zhejiang Food Safety Expert Committee, director of Zhejiang Food Science and Technology Society, and member of the Standing Committee of Hangzhou Association for Science and Technology , Deputy Director of Hangzhou Food and Drug Safety Emergency Expert Committee, Chairman of Hangzhou Food and Nutrition Society, Food Science and Human Wellness , BioFactors , Food and-Nutritional Science , Annals of Nutrition and Food Science , International Journal of Trends and Technologies in Food Processing , Nutrition_Dietetics , Vegetarian Nutrition Journal and other international journal editorial boards, as well as Cancer , Annals of Clinical Micro Journal of Nutrition and Metabolism , Experimental Gerontology ,Reviewer for more than 30 international journals including Journal of Intercultural Ethnopharmacology , Acta Biochimica et Biophysica , Plant Foods for Human Nutrition , Journal of Food & NutritionalDisorders , Journal of Zhejiang University-SCIENCE B , International Journal of Insect Science
, e.

Professor Shen Lirong has been engaged in the basic research and application development of food nutrition health and food safety for a long time, especially in the fields of nutrition and health function of royal jelly, honey, camellia oil, and turmeric, and has made outstanding achievements in the field of quality testing technolo.

.

The main research directions are the selection of new varieties of pear and jujube, and the physiology and technology of fruit quali.

Comparative transcriptome analysis on candidate genes involved in lipid biosynthesis of developing kernels for three walnut cultivars in Xinjiang

Wenqiang Wang ^a , Hao Wen ^a , Qiang Jin ^b , Wenjun Yu ^b , Gen Li ^b , Minyu Wu ^a , Hongjin Bai ^b , Lirong Shen ^a,* , Cuiyun Wu ^b,*

^a Department of Food Science and Nutrition, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou 310058, China

^b College of Horticulture and Forestry, Tarim University, Alar 843300, China

*Corresponding autho.

E-mail address: shenlirong@z.

wcyby@16com

Abstract

Walnut ( Juglans regi.