Construction and analysis of cotton (Gossypium arboreum L.) drought-related cDNA library

Abstract

Background: Drought is one of the most important environmental factors causing water stress for cotton, and it greatly limits cotton growth and crop productivity. So far only a few drought-tolerance genes have been functionally characterized in details, and most efforts on this topic have been made in model organisms. Therefore, to identify more drought-related genes in cotton plays a crucial role in elucidating the underlying mechanisms of drought tolerance as well as utilizing bioengineering techniques to improve the tolerance in this organism.

Findings: Here we constructed a subtractive drought-tolerance cDNA library using suppressive subtractive hybridization (SSH). Through differential screening and bioinformatics analysis, we identified 392 positive clones with differential expression, corresponding 265 unique genes. By BLAST search against Genbank, we found that more than half of these EST sequences were homologous to those previously known drought-related genes and that there were 57 sequences with unknown functions, suggesting that many more genes are involved in this complex trait. Moreover, using RT-PCR, we examined the expression of nine representative candidate genes and confirmed that their expression levels were increased at different levels under drought stress.

Conclusion: Our results show that drought tolerance is a complex trait in cotton, which involves the coordination of many genes and multiple metabolism pathways. The candidate EST sequences we identified here would facilitate further functional studies of drought-related genes and provide important insights into the molecular mechanisms of drought-stress tolerance and genetic breeding in cotton.

Figure 1

The analysis on the SSH library. Analysis of ds cDNA synthesis products and Rsa I digestion. Cotton double-stranded cDNA before (Lane 2) and after (Lane 1) Rsa I digestion. Lane M: DNA size markers. B) Analysis of ligation efficiency. Lane 1: PCR products using Tester 1-1 (Adaptor 1-ligated) as the template and the Histone 3 3' primer and PCR primer 1. Lane 2: PCR products using Tester 1-1 (Adaptor 1-ligated) as the template, and the Histone 3 3' and 5' primers. Lane 3: PCR products using Tester 1–2 (Adaptor 2R-ligated) as the template, and the Histone 3 3' primer and PCR primer 1. Lane 4: PCR products using Tester 1–2 (Adaptor 2R-ligated) as the template, and the Histone 3 3' and 5' primers. Lane M: DNA size markers. C) Results of PCR-select cDNA subtraction analysis. Lane 1: Forward secondary PCR products of unsubtracted. Lane 2: Forward secondary PCR products of subtracted. Lane 3: Reverse secondary PCR products of unsubtracted. Lane 4: Reverse secondary PCR products of subtracted. Lane M: DNA size markers. D) Analysis of subtraction efficiency using PCR. The subtracted and unsubtracted pools of cDNA were amplified by using primers for the constitutively expressed Histone 3 gene. PCR was performed on the subtracted (Lanes 1–4) or unsubtracted (Lanes 5–8) secondary PCR product with the Histone 3 5' and 3' primers. Lanes 1 & 5: 18 cycles; Lanes 2 & 6: 23 cycles; Lanes 3 & 7: 28 cycles; Lanes 4 & 8: 33 cycles.

Figure 2

The PCR analysis of partial clones in the subtracted library. Lane 1–24: PCR products from different clones. Lane M: DNA size markers.

Figure 3

The GO classification of cotton drought-tolerance genes. The bar charts show the distribution of ESTs among three principal GO categories: cellular location, molecular function and biological process.

Figure 4

The COG categories of drought-tolerance gene in cotton. The drought-related gene sets differ in their distribution of COG categories (E-value < 1e-05). The pie chart is color-coded as per COG colors with the COG functional annotation, and represented the percentages of genes per COG category.

Figure 5

The RT-PCR expression analysis of cotton nine clones isolated by SSH. Gene-specific primers for nine clones were used to amplify a cDNA fragment of the corresponding gene after total RNA was reversely transcribed from drought-stressed cotton seedlings by 17% PEG6000. 1–6: The different time point from 1.5 h to 6.5 h. Histone 3 was used to normalize the amount of template in the PCR reactions.