CGPDB - Description of Library Construction

Plant material

            Two distinct lettuce genotypes were used for the construction of lettuce cDNA libraries, cultivated lettuce (Lactuca sativa L. cv. Salinas) and wild lettuce (Lactuca serriola L.).  The following tissues were collected from both genotypes: callus, roots, environmentally stressed roots, pre-fertilized flowers, post-fertilized flowers, environmentally stressed flowers, chemically induced seedlings, environmentally stressed shoots, germinating seeds, and dark grown leaves.

            The sunflower tissues were collected from Helianthus annuus lines RHA801 and RHA280, Helianthus paradoxus, and Helianthus argophyllus. Eleven different tissues from both genotypes of Helianthus annuus were used: callus, roots, disk and ray flowers, pre-fertilized flowers, developing kernel, chemically treated seedlings, environmentally stressed roots, environmentally stressed shoots, germinating seeds, environmentally stressed flowers, and hulls.

            Salt stressed and normal seedlings were collected from Helianthus paradoxus, and drought stressed and normal seedlings were collected from Helianthus argophyllus.  All plant tissues were frozen in liquid nitrogen upon collection and stored at –80C.

 

Library construction

            The plant tissues were ground in liquid nitrogen and resuspended in 1:1 mixture of RNA extraction buffer (0.1 M Tris-HCl, pH 9.0, 0.1 M LiCl, 10 mM EDTA, and 1% [w / v] SDS) and saturated phenol at 70C.  After vortexing and centrifugation (15 min, 6,000 g), the water phase was collected and RNA extracted as described by Pawlowski et al. (1994).

            Standard EST libraries were made using a modified SMARTTM (BD Clontech, Palo Alto, CA) approach. The tissue samples were tagged during cDNAs synthesis using the tissue assigned primers.  Each cDNA was made using oligonucleotide primers that incorporated unique 5' sequence tags so that the source of each sequence could be subsequently identified (Table 1, Figure 1).  For each genotype (both lettuce species and from sunflower lines RHA801 and RHA280), the cDNA samples were pooled (click here to open pooling scheme in a new browser window) after the second PCR step, size fractionated in agarose gels into four size classes.  Each size class (0.2 – 0.8 kb, 0.8 – 1.5 kb, 1.5 – 3 kb, and > 3 kb) was directionally cloned into a medium copy vector pBRcDNASfiIAB and transformed separately to reduce size bias.

            The PCR-Select Subtraction Kit (BD Clontech, Palo Alto, CA) was used to make Helianthus paradoxus salt induced and salt repressed libraries, and Helianthus argophyllus drought induced and drought repressed libraries.  This protocol uses suppression PCR to enrich for transcripts differentially expressed in the tested samples (Diatchenko et al., 1996).

            For the salt induced library, we performed the subtractive hybridization using H. paradoxus salt induced sample as tester, and H. paradoxus normal tissue as driver sample.  For the construction of salt repressed library the subtraction was in the reverse direction, so the normal tissue was used as tester, and the salt treated one was driver.  For the drought induced library, the Helianthus argophyllus drought treated sample was used as tester, and H. agrophillus normal sample was driver, and the subtraction direction was reversed during construction of the drought-repressed H. agrophillus library.  Following PCR amplification, subtracted cDNAs were cloned into pGEM vector using the TA cloning system from Promega (Madison, WI).  Recombinant clones were identified on X-Gal-containing plates.  The average insert size was around 300 bp, resulting from the cDNA restriction step incorporated into the subtraction protocol.

            Summary of cloning and sequencing per library is shown on Table 2  (this web page) and Table 3 (open Table 3 in a new browser window).



Table 1.
TAG sequences (adaptors).  Sfi restriction sites are indicated in italic.  Tagging sites are underlined

TAG ID

sequence

lettuce tissue/treatment

sunflower tissue/treatment

TAG0

GGCCATTATGGCC GTACTG CGGG

callus

callus

TAG1

GGCCATTATGGCC GTTGCA CGGG

root

root

TAG2

GGCCATTATGGCC ACATGT CGGG

not in use

disk and ray flowers

TAG3

GGCCATTATGGCC GCTTGA CGGG

flowers pre-fertilized

flowers pre-fertilized

TAG4

GGCCATTATGGCC TGCCAT CGGG

flowers post-fertilized

developing kernel

TAG5

GGCCATTATGGCC TGTAGC CGGG

chemical induction

chemical induction

TAG6

GGCCATTATGGCC TTGCTC CGGG

not in use

not in use

TAG7

GGCCATTATGGCC ATCTCG CGGG

roots environmental stress

roots environmental stress

TAG8

GGCCATTATGGCC TCGCAA CGGG

shoots environmental stress

shoots environmental stress

TAG9

GGCCATTATGGCC TCTGTG CGGG

germinating seeds

germinating seeds

TAG10

GGCCATTATGGCC CGAATG CGGG

flowers environmental stress

flowers environmental stress

TAG11

GGCCATTATGGCC GCTAGT CGGG

leaves dark grow

hulls



Figure 1. Cloning of cDNA fragments using pBRcDNA-sfi-AB Vector


cDNA Cloning


Table 2.
Number of EST reads per library


 

genotype

library
name

tissue and
treatment

# of ESTs

lettuce

Lactuca sativa,
cv.
Salinas

QG_ABCDI

10 different sources of RNA
(details in Table 3)

32,881

Lactuca serriola

QG_EFGHJ

10 different sources of RNA
(details in Table 3)

35,316

sunflower

Helianthus annuus,
line RHA801

QH_ABCBI

11 different sources of RNA
(details in Table 3)

22,920

Helianthus annuus,
line RHA280

QH_EFGHJ

11 different sources of RNA
(details in Table 3)

21,133

Helianthus paradoxus

QH_K

seedling, root, leaf and flower
(normal conditions)

4,391

Helianthus paradoxus

QH_L

seedling, root, leaf and flower
(salt stress)

5,949

Helianthus argophyllus

QH_M

seedling, root and leaf
(normal conditions)

5,483

Helianthus argophyllus

QH_N

seedling, root and leaf
(drought stress)

7,304




Download sequence of pBRcDNASfiIAB vector
Download oligo sequences Primers_pBRcDNASfiIAB

Literature cited

Pawlowski K, Kunze R, de Vries S, Bisseling T (1994) Isolation of total, poly (A) and polysomal RNA from plant tissues.  In SB Gelvin, RA Schilperoort, eds, Plant Molecular Biology Manual. Kluwer Academic Publishers, Norwell, MA, pp 1-13


email:
Alexander Kozik akozik@atgc.org
Richard Michelmore michelmore@vegmail.ucdavis.edu

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CGPDB
Compositae Genome Project

last modified: January 22 2004